MEDICAL 


Gift   of 


Panama-Pacific  Intern 
Exposition  Company 


PATHOGENIC  MICRO-ORGANISMS 


M  AC  N  E  A  L 


PATHOGENIC 
MICRO-ORGANISMS 

A  TEXT-BOOK  OF 

MICROBIOLOGY   FOR   PHYSICIANS 
AND  STUDENTS  OF  MEDICINE 


BY 

WARD  J.  IV^ACNEAL,  PH.  D.,  M.  D., 

PROFESSOR  OF  PATHOLOGY  AND  BACTERIOLOGY  IN  THE  NEW  YORK 
POST-GRADUATE  MEDICAL  SCHOOL  AND   HOSPITAL,   NEW  YORK 


(Based  Upon  Williams'  Bacteriology) 


WITH  213  ILLUSTRATIONS 


PHILADELPHIA 

P.   BLAKISTON'S  SON   &   CO. 

1012  WALNUT  STREET 
1914 


COMPLIMENTS 
OF 


sion's  Son  Co. 


COPYRIGHT,  1914,  BY  P.  BLAKISTON'S  SON  &  Co. 


THE- MAPLE- PRESS. YORK. PA 


PUBLISHER'S  ANNOUNCEMENT 

When  Professor  H.  U.  Williams  was  requested  to  undertake 
the  sixth  revision  of  this  book  he  expressed  a  wish  to  be  relieved 
of  it  and  of  all  further  obligations  in  respect  thereto  because  oi 
the  demands  that  were  being  made  upon  his  time  by  largei 
duties  in  connection  with  the  University. 

As  this  manual  is  a  popular  one  we  did  not  feel  warranted  in 
letting  it  go  out  of  print  as  suggested  by  Dr.  Williams,  therefore 
we  arranged  with  Professor  Ward  J.  MacNeal  to  continue  it  with 
the  approval  of  Professor  Williams.  The  wisdom  of  this  choice 
should  be  evident  in  the  following  pages. 


100 


PREFACE. 

This  volume  is  the  outgrowth  of  an  attempt  to  revise  the  well- 
known  William's  Manual  of  Bacteriology,  undertaken  at  the  invi- 
tation of  the  Publishers,  Messrs.  P.  Blakiston's  Son  and  Co.,  very 
cordially  seconded  by  Dr.  Williams,  who  kindly  placed  the  mate- 
rial of  the  previous  editions  at  my  disposal.  The  text  has  been 
very  largely  rewritten  and  the  order  of  treatment  considerably 
altered.  Many  of  the  illustrations  of  Dr.  Williams  have  been 
retained  and,  as  they  have  not  been  acknowledged  in  the  legends,  I 
wish  to  express  my  special  obligation  for  them  in  this  place. 

The  book  is  intended  as  an  introduction  to  the  study  of  patho- 
genic micro-organisms  and  is  designed  especially  for  the  use  of 
physicians  and  students  of  medicine.  During  the  past  decade, 
the  parasitic  protozoa  have  assumed  an  importance  which  places 
them  almost  on  a  par  with  the  bacteria  as  pathogenic  agents,  and 
the  extension  of  bacteriological  methods  to  the  study  of  molds, 
yeasts,  filterable  viruses  and  protozoa  has  tended  again  to  re- 
unite the  various  portions  of  this  field  of  knowledge,  much  as  it 
was  in  the  days  of  Pasteur.  The  attempt  has  here  been  made  to 
outline  the  subject  and  to  present  a  few  examples  under  each 
important  heading,  in  the  hope  that  the  student  may  become 
acquainted  with  the  broad  principles  of  the  science  and  appreciate 
the  variety  of  procedures,  conceptions  and  organisms  with  which 
it  deals.  Part  I  is  devoted  to  a  description  of  technical  procedures, 
Part  II  to  the  general  biology  of  micro-organisms  and  Part  III 
to  a  consideration  of  individual  microbes.  Much  has  of  necessity 
been  omitted  and  many  topics  treated  only  very  briefly. 

In  the  preparation  of  the  manuscript  considerable  use  has 
been  made  of  the  text-books  of  McFarland,  Jordan,  Marshall, 
and  of  Hiss  and  Zinsser,  and  the  Handbuch  der  pathogenen  Mikro- 
organismen  of  Kolle  and  Wassermann  and  Doflein's  Lehrbuch  der 

vii 


Vlll  PREFACE 

Protozoenkunde  have  been  most  extensively  employed.  Numer- 
ous illustrations  have  been  copied  from  the  last  mentioned  work 
and  the  text  itself  has  been  closely  followed  in  many  places. 

The  attempt  has  been  made  to  give  proper  credit  for  borrowed 
illustrations,  but  the  numerous  cuts  retained  from  the  previous 
editions  of  Williams  have  not  been  specially  designated.  Some 
references  have  been  included,  to  offer  the  student  a  ready  intro- 
duction to  the  literature  of  the  topic  under  discussion,  especially 
in  those  instances  in  which  the  topic  has  been  only  briefly  men- 
tioned here. 

My  thanks  are  due  to  the  Press  of  Gustav  Fischer,  Jena,  for 
the  loan  of  numerous  illustrations,  to  P.  Blakiston's  Son  and 
Company  for  their  uniform  courtesy  in  our  relations,  and  espe- 
cially to  my  wife  whose  enthusiastic  assistance  has  made  possible 
the  preparation  of  the  manuscript  and  has  lightened  the  burden 
of  seeing  it  through  the  press. 

W.    J.    MACNEAL. 
NEW  YORK 
December,  1913. 


TABLE  OF  CONTENTS. 

INTRODUCTION. 

Bacteriology  and  Microbiologys,  i ;  Biological  relationship,  3 ;  Spontaneous  genera- 
tion, 3;  Heterogenesis,  4;  Systematic  relationships,  4;  Fermentation  and  Putre- 
faction, 5;  Specific  fermentations,  6;  Pathology  and  Hygiene,  7;  Contagion,  8; 
Specific  infection,  10;  Antisepsis,  n;  Proof  of  the  germ  theory,  n;  Immunity, 
12;  Parasitic  protozoa,  12;  Insect  transmission,  13;  Pathogenic  spirochetes,  13; 
Filterable  viruses,  13;  Agriculture,  14;  Biological  view-point  in  the  study  of 
micro-organisms,  14. 

PART  I.     BACTERIOLOGICAL  TECHNIC. 
CHAPTER  I. — THE    MICROSCOPE    AND    MICROSCOPIC    METHODS. 

Development  of  the  microscope,  15;  Lenses,  15;  Achromatic  and  apochromatic 
objectives,  15;  Ultra- microscope  and  dark-field  microscopy,  16;  Tandem  micro- 
scope, 16;  Principle  of  the  microscope,  16;  Pin-point  aperture,  16;  Relations  of 
magnification,  definition  and  brilliancy  of  image,  16;  Lens-armed  aperture,  17; 
Two  lenses  in  series,  18;  Magnification  measured  by  the  ratio  of  the  opening 
and  closing  angles  of  a  beam,  19;  Simple  microscope,  19;  Reading  glass,  19; 
Spherical  aberration,  20;  Chromatic  aberration,  20;  Diffraction,  20;  Image 
formation  in  compound  microscope,  22;  Numerical  aperture,  23;  Illumination 
by  the  Abbe  condenser,  24;  Central  illumination,  24;  Dark-field,  25;  Illumina- 
tion by  broad  converging  beam,  25;  Visibility  of  microscopic  objects,  25 ;  Defini- 
tion by  light  and  shade,  26;  The  color  picture,  28;  The  Bacteriological  micro- 
scope, 29 ;  Eye-pieces  and  objectives,  30;  Use  of  the  microscope,  31;  Microscopic 
measurements,  31;  The  platinum  wire,  31 ;  Pasteur  pipettes,  32 ;  The  hanging- 
drop,  33 ;  Motility  of  micro-organisms,  34;  Brownian  motion,  34;  Hanging- 
block,  35 ;  Slide  for  dark-field  study,  35 ;  Use  of  dark-field,  36;  Smear  prepara- 
tions, 36 ;  Cover-glasses,  36;  Technic,  37;  Slide  smears,  39;  Staining  solutions,  39 ; 
Aniline  stains,  40;  Method  of  simple  staining,  44;  Gram's  stain,  44;  Acid-proof 
staining,  46;  Sputum  staining,  47;  Spore  staining,  50;  Capsule  stain,  51 ;  Stain- 
ing of  flagella,  52 ;  Wet  fixation,  54 ;  Iron  hematoxylin,  54 ;  Blood  films,  54 ; 
Staining  of  tissue  sections,  55;  Celloidin,  55;  Paraffin,  55;  Sectioning,  56; 
Simple  staining,  58;  Gram-Weigert  method,  59;  Tubercle  bacilli,  60;  Nuclear 
stains,  61. 

ix 


X  TABLE  OF  CONTENTS 

CHAPTER  II.     STERILIZATION,  DISINFECTION,  ANTISEPSIS,  FOOD 
PRESERVATION. 

Definitions,  62 ;  Physical  sterilization,  62 ;  Mechanical  removal,  62;  Desiccation,  63; 
Light,  63;  Cold,  64;  Heat,  64;  Electricity,  71;  Chemical  sterilization,  71 ;  Soaps, 
71;  Acids,  71;  Alkalies,  73;  Oxidizing  agents,  73;  Inorganic  salts,  74;  Organic 
poisons,  76;  Antiseptics  and  preservatives,  79;  Physical,  79;  Chemical,  79; 
Testing  of  antiseptics  and  disinfectants,  80. 

CHAPTER  III.     CULTURE  MEDIA. 

Definition,  83  ;  Glass-ware,  83 ;  The  common  media,  84 ;  Nutrient  broth,  84;  Titra- 
tion  of  media,  85;  Gelatin,  88;  Agar,  89;  Modifications,  90;  Sterilizable  special 
media,  91 ;  Potato,  91;  Milk,  92;  Peptone  solution,  92;  Nitrate  broth,  92;  Blood- 
serum,  92;  Loeffler's  blood-serum,  94;  Eggs,  94;  Dorset's  egg,  94;  Bread  paste, 
94;  Media  containing  uncooked  protein,  95;  Sterile  blood,  95;  Ascitic  fluid,  97; 
Sterilization,  97;  Sterile  tissue,  98;  Blood-streaked  agar,  98;  Blood-agar,  98; 
Broth  containing  tissues,  99;  Ascitic-fluid  agar,  99;  Ascitic  fluid  with  tissue,  99. 

CHAPTER    IV.     COLLECTION    OF    MATERIAL    FOR    BACTERIOLOGICAL 

STUDY. 

General  considerations,  100;  Sampling  water  and  foods,  100;  Material  from  the 
body,  100 ;  Sputum,  101;  Urine,  101;  Blood  and  transudates,  101;  Cerebro-spinal 
fluid.  101;  Feces  and  intestinal  juice,  102;  Pus  and  exudates,  102;  Material  from 
autopsies,  103. 

CHAPTER  V.    THE  CULTIVATION  OF  MICRO-ORGANISMS. 

Avoidance  of  contamination,  104;  Isolation  of  bacteria,  105;  Plate  cultures,  106; 
Roll  tubes,  no;  Streak  method,  112;  Tall-tube  method,  112;  Colonies,  113; 
Pure  cultures,  113;  Stock  cultures,  114;  Regulation  of  temperature,  115;  High 
temperature  incubator,  115;  Gas-regulator,  116;  Automatic  safety-burner,  120; 
Incubator  room,  120;  Prevention  of  drying,  120;  Low-temperature  incubator, 
121;  Cultivation  of  anaerobic  bacteria,  124;  Deep  stab,  124;  Veillon  tall-tube 
method,  124;  Fermentation  tube,  125;  Removal  of  oxygen,  125;  Hydrogen  at- 
mosphere, 126;  Further  methods,  130. 

CHAPTER  VI.     METHODS  OF  ANIMAL  EXPERIMENTATION. 

Value  of  Animal  experimentation,  131 ;  Care  of  animals,  131 ;  Holding  for  operation, 
132;  Inoculation,  133;  Subcutaneous  and  intraperitoneal,  133;  Intracranial, 
133;  Into  circulating  blood,  133;  Other  sites,  134;  Subcutaneous  application, 
134;  Alimentary  and  respiratory  infection,  134;  Collodion  capsules,  134;  Obser- 
vation of  infected  animals,  136. 


TABLE  OF  CONTENTS  XI 


PART  II.     GENERAL  BIOLOGY  OF  MICRO-ORGANISMS. 

CHAPTER  VII.    MORPHOLOGY  AND  CLASSIFICATION. 

Molds,  137 ;  Yeasts,  140 ;  Bacteria,  141 ;  Trichobacteria,  141;  Spherical  bacteria,  142; 
Cylindrical  bacteria,  144;  Spiral  bacteria,  146;  Structure  of  the  lower  bacteria, 
147;  Endospores,  149;  Filterable  viruses,  150;  Protozoa,  150;  Flagellates,  151; 
Rhizopods,  154;  Sporozoa,  155;  Ciliates,  159;  Outline  classification  of  micro- 
organisms, 160 ;  Specific  nomenclature,  160. 

CHAPTER  VIII.     PHYSIOLOGY  or  MICRO-ORGANISMS. 

Relations  of  morphology  and  physiology,  162 ;  Conditions  of  physiological  study,  163 ; 
Environmental  factors,  164;  Moisture,  164;  Organic  food,  164;  Inorganic  salts 
and  chemical  reaction,  165;  Oxygen,  166;  Temperature,  166;  Microbic  variation, 
167;  Products  of  microbic  growth,  168;  Physical  effects,  168;  Chemical  effects, 
168;  Enzymes,  169;  Toxins,  171;  Relation  of  microbe  and  its  environment,  171 ; 
Morphological  characters,  171;  Physiological  tests,  173;  Descriptive  chart  of 
the  Society  of  American  Bacteriologists,  173. 

CHAPTER  IX.    THE  DISTRIBUTION  or  MICRO-ORGANISMS  AND  THEIR 
RELATION  TO  SPECIAL  HABITATS. 

General  distribution,  174;  Micro-organisms  of  the  Soil,  175;  Pathogenic  soil 
bacteria,  176;  Micro-organisms  of  the  air,  176;  Micro-organisms  of  Water  and 
Ice,  178;  Self-purification  of  water,  179;  Storage  of  water,  180;  Filtration,  180; 
Disinfection  of  water,  182;  Bacteriological  examination  of  water,  182;  Detec- 
tion of  intestinal  bacteria,  186;  Bacteriological  examination  of  ice,  188;  Micro- 
organisms of  food,  189;  Milk,  189;  Milk  flora,  190;  Pathogenic  microbes  in 
milk,  192;  Milk  for  infant  feeding,  192;  Other  foods,  193. 

CHAPTER  X.     PARASITISM  AND  PATHOGENESIS. 

The  parasitic  relation,  194;  Pathogenesis,  195;  Rules  of  Koch,  195;  Infectious 
disease,  196;  Possibility  of  infection,  196;  Susceptibility  and  resistance,  196; 
Number  of  invaders,  197;  Modes  of  introduction,  197;  Local  susceptibility,  199; 
Local  and  general  infections,  199;  Transmission  of  infection,  200;  Healthy 
carriers  of  infection,  201. 

CHAPTER  XI.    THE  PATHOGENIC  PROPERTY  or  MICRO-ORGANISMS. 

Adaptation  to  parasitism,  202 ;  Virulence,  202 ;  Microbic  poisons,  203 ;  Defensive 
mechanisms,  204. 


Xll  TABLE  OF  CONTENTS 

CHAPTER  XII.     REACTION  or  THE  HOST  TO  INFECTION. 

Facts  and  theories,  206 ;  Physiological  hyperplasia,  206 ;  Phagocytosis  and  encapsu- 
lation, 207;  Chemical  constitution  of  the  cell,  207;  Antitoxins,  208;  Cell 
receptor  of  first  order,  209;  Precipitins,  209;  Receptor  of  second  order,  210; 
Agglutinins,  211;  Phenomenon  of  agglutination,  211;  Bactericidal  substances, 
212;  Cytolysins,  213;  Receptor  of  third  order,  214;  Amboceptor  and  comple- 
ment, 214;  Deviation  of  complement,  215;  Fixation  of  complement,  216; 
Opsonins,  217;  Anti-aggressins,  218;  Source  and  distribution  of  antibodies, 
218;  Allergy,  219. 

CHAPTER  XIII.     IMMUNITY  AND  HYPERSUSCEPTIBILITY.    THEORIES 

or  IMMUNITY. 

Immunity,  220;  Natural  immunity,  220;  Immunity  of  species,  220.  Racial  im- 
munity, 221;  Individual  variations,  221;  Acquired  immunity,  222;  Active 
immunity,  222.  Passive  immunity,  224.  Combined  active  and  passive 
immunity,  225  ;  Mechanisms  of  immunity,  225 ;  Hypersusceptibility  or  Ana- 
phylaxis,  226 ;  Theories  of  immunity,  227. 

PART  III.     SPECIFIC  MICRO-ORGANISMS. 

CHAPTER  XIV.     THE  MOLDS  AND  YEASTS  AND  DISEASES  CAUSED  BY 

THEM. 

Mucors,  231;  Aspergilli,  233;  Penicillium  crustaceum,  234;  Claviceps  purpurea, 
234;  Ergotism,  235;  Botrytis  bassiana,  235;  Muscardine,  236;  Oidium  lactis, 
236;  Oidium  albicans,  238;  Thrush,  238;  Achorion  schoenleinii,  239;  Favus, 
239;  Microsporon  audouini,  241;  Alopecia  areata,  241;  Microsporon  furfur, 
242 ;  Tricophyton  acuminatum,  242 ;  Sporotrichum  schenki,  242 ;  Sporotri- 
chum  beurmanni,  244;  Saccharomyces  cerevisiae,  244;  Blastomycetic  derma- 
titis, 244 ;  Coccidioidal  granuloma,  245. 

CHAPTER  XV.     TRICHOMYCETES. 

Actinomyces  bovis,  246;  Streptothrax  madurae,  248;  Mycetoma,  248;  Cladothrix, 
249 ;  Leptothrix  buccalis,  249. 

CHAPTER  XVI.    THE  COCCACE^E  AND  THEIR  PARASITIC 
RELATIONSHIPS. 

Diplococcus  gonorrheae,  250;  Occurrence,  250.,  Culture,  250;  Toxins,  253;  Gonor- 
rhea, 252;  Specific  diagnosis,  253;  Prophylaxis,  253;  Diplococcus  meningi- 
tidis,  253;  Anti-meningococcus  serum,  255;  Quincke's  puncture,  255;  Exami- 


TABLE  OF  CONTENTS  Xlll 

nation  of  spinal  fluid,  256;  Diagnosis,  257;  Diplococcus  catarrhalis,  257; 
Diplococcus  pneumonias,  257;  Occurrence,  257;  Morphology,  258;  Cultures, 
258;  Pneumonia,  259;  Toxins,  259;  Immunity,  260;  Streptococcus  viridans, 
260 ;  Streptococcus  mucosus,  260 ;  Streptococcus  pyogenes,  260 ;  Occurrence, 
261;  Cultures,  261;  Animal  inoculation,  262;  Surgical  infections,  263;  Erysip- 
elas, 263;  Puerperal  fever,  263;  Immunity,  264;  Streptococcus  lacticus,  264 ; 
Staphylococcus  aureus,  264;  Occurrence,  264.  Morphology,  264;  Cultures, 
265;  Toxins,  265;  Animal  inoculation,  266;  Infection  of  man,  266;  Immunity, 
266;  Vaccine  therapy,  266;  Staphylococcus  alb  us,  267;  Micrococcus  tetra- 
genus,  267;  Sarcina  ventriculi,  267;  Sarcina  aurantiaca,  267;  Micrococcus 
agilis,  267. 

CHAPTER  XVII.     B ACTERIACE^E  :  THE  SPOROGENIC  AEROBES. 

Bacillus  mycoides,  268 ;  Bacillus  vulgatus,  268 ;  Bacillus  subtilis,  269 ;  Parasitism 
269;  Bacillus  anthracis,  270;  Occurrence,  270;  Morphology,  271;  Resistance 
272;  Anthrax,  272;  Human  anthrax,  273;  Immunity,  273;  Seium,  274. 

CHAPTER  XVIII.     BACTERIACE.E:  THE  SPOROGENIC  ANAEROBES. 

Group  characters  and  habitat,  275 ;  Bacillus  endematis,  275 ;  Putrefactive  prop- 
erties, 276;  Malignant  edema,  276;  Bacillus  feseri,  276;  Bacillus  welchii, 
276;  Occurrence,  276;  Characters,  277;  Emphysematous  gangrene,  277;  Bac- 
illus tetani,  278;  Occurrence,  278;  Morphology,  278;  Cultures,  279;  Toxin, 
279;  Tetanus,  280;  Immunity,  281;  Antitoxin,  281;  Standard  unit,  282;  Pro- 
phylaxis and  treatment,  283;  Bacillus  botulinus,  283 ;  Botulin,  284;  Immune 
serum,  284;  Botulism,  284. 

CHAPTER  XIX.     BACTERIACE^E:  THE  BACILLUS  or  DIPHTHERIA  AND 

OTHER  SPECIFIC  BACILLI  PARASITIC  ON  SUPERFICIAL  Mucous 

MEMBRANES. 

Bacillus  diphtherias,  285;  Occurrence,  285;  Culture,  285;  Toxin,  288;  Diphtheria, 
289;  Bacteriological  diagnosis,  290;  Transmission  of  the  disease,  292;  Immu- 
nity, 292;  Antitoxin,  293;  Standard  unit  of  antitoxin,  294;  Prophylactic  and 
therapeutic  use  of  antitoxic  serum,  295;  Untoward  effects,  295;  Bacillus 
xerosis,  295;  Bacillus  hoffmanni,  296;  Morax-Axenfeld  bacillus,  296;  Koch- 
Weeks  bacillus,  296 ;  Bacillus  pertussis,  296 ;  Bacillus  influenzas,  297 ;  Bac- 
illus chancri,  298. 

CHAPTER  XX.     BACTERIACE.E:  THE  TUBERCLE  BACILLUS  AND  OTHER 
ACID-PROOF  BACTERIA. 

Bacillus  tuberculosis,  299;  Human  type,  300;  Occurrence,  300;  Morphology,  300; 
Cultures,  301;  Chemical  composition,  302;  Toxins,  303;  Resistance,  304;  Tuber- 


XIV  TABLE  OF  CONTENTS 

culin,  304;  Animal  inoculation,  305;  Tuberculosis,  305;  The  Tubercle,  306; 
Mode  of  transmission,  307;  Bacteriological  diagnosis,  307;  Allergic  reactions, 
308;  Bovine  type,  310;  Avian  type,  311;  Fish  or  amphibian  type,  312;  Bac- 
illus leprae,  312;  Morphology  and  occurrence,  312;  Leprosy,  313;  Bacillus 
smegmatis,  313;  Bacillus  moelleri,  314;  Other  acid-proof  organisms,  314; 
Pseudo-bacilli,  315. 

CHAPTER  XXI.     B  ACTERIACE^E  :  THE  BACTERIA  OF  THE  HEMOR- 
RHAGIC  SEPTICAEMIAS,  OF  PLAGUE  AND  OF  MALTA  FEVER. 

Bacillus  avisepticus,  316;  Bacillus  plurisepticus,  316;  Bacillus  pestis,  317;  Occur- 
rence and  morphology,  317;  Cultures,  318;  Toxins,  318;  Animal  inoculation, 
319;  Bubonic  plague,  319;  Epizootic  plague,  320;  Human  plague,  320;  Immu- 
nity, 320;  Immune  serum,  321;  Prophylaxis,  321;  Eradication  of  endemic 
centers,  321;  Bacillus  melitensis,  321;  Malta  fever,  322. 

CHAPTER  XXII.     BACTERIACE^E:  THE  COLON,  TYPHOID  AND 
DYSENTERY  BACILLI. 

Bacillus  coli,  324;  Occurrence  and  morphology,  324;  Cultures,  325;  Pathogenic 
relations,  325;  Bacillus  aerogenes,  326;  Bacillus  pneumoniae,  327;  Bacillus 
rhinoscleromatis,  327;  Bacillus  enteritidis,  328;  Bacillus  suipestifer,  329; 
Bacillus  psittacosis,  329 ;  Bacillus  typhi  murium,  329 ;  Bacillus  alkaligenes, 
329;  Bacillus  typhosus,  330;  Occurrence  and  morphology,  330;  Cultures,  331; 
Resistance,  332;  Toxins,  332;  Animal  inoculation,  332;  Typhoid  fever,  333; 
Bacteriological  diagnosis,  333;  Transmission  of  the  disease,  334;  Prevention, 
335;  Bacillus  dysenteriae,  336;  Epidemic  dysentery,  336;  Paradysentery 
bacilli,  337. 

CHAPTER  XXIII.     B ACTERIACE^E  :  BACILLUS  MALLEI  AND  MISCEL- 
LANEOUS BACILLI. 

Bacillus  mallei,  339;  Occurrence  and  morphology,  339;  Cultures,  339;  Mallein, 
340;  Glanders,  340;  Bacteriological  diagnosis,  340;  Bacillus  abortus,  341 ; 
Bacillus  acne,  342;  Bacillus  bifidus,  342;  Bacillus  bulgaricus,  342;  Bacillus 
vulgaris,  343;  Bacillus  pyocyaneus,  343;  Bacillus  fluorescens,  343;  Bacillus 
violaceus,  343;  Bacillus  cyanogenus,  343;  Bacillus  prodigiosus,  344. 

CHAPTER  XXIV.     SPIRILLACE.E    AND    THE    DISEASES    CAUSED    BY 

THEM. 

Spirillum  rubrum,  345 ;  Spirillum  choleras,  345 ;  Occurrence  and  morphology,  345 ; 
Cultures,  345;  Animal  inoculation,  347;  Toxins,  348;  Pfeiffer's  phenomenon, 
348;  Asiatic  cholera,  348;  Mode  of  infection,  349;  Bacteriological  diagnosis, 
350;  Prophylaxis,  351;  Spirillum  metchnikovi,  352;  Spirillum  Finkler -Prior, 
352 ;  Spirillum  tyrogenum,  352. 


TABLE  OF  CONTENTS  XV 


CHAPTER  XV. 

Spirochseta  plicatilis,  353;  Other  saprophytic  spirochetes,  353;  Spirochaeta  recur- 
rentis,  353;  Varieties,  354;  Cultures,  354;  Diagnosis  of  relapsing  fever,  355; 
Spirochaeta  anserina,  356;  Spirochaeta  gallinarum,  356;  Spirochaeta  muris, 
356;  Spirochaeta  pallida,  357;  Occurrence  and  morphology,  357;  Cultures, 
358;  Luetin,  359;  Syphilis,  360;  Bacteriological  diagnosis,  360;  Microscopic 
detection  of  spirochetes,  360;  Animal  inoculation,  361;  Wassermann  reaction, 
361;  Luetin  test,  366;  Spirochaeta  refringens,  366;  Spirochaeta  microdentium, 
366 ;  Spirochaeta  (Bacillus)  fusiformis,  367. 

CHAPTER  XXVI.     THE  FILTERABLE  MICROBES. 

The  virus  of  foot-and-mouth  disease,  368 ;  The  virus  of  bovine  pleuro-pneumonia, 
368;  The  virus  of  yellow  fever,  368;  Occurrence  and  nitration,  368;  Yellow 
fever,  369,  Transmission,  369;  Prophylaxis;  369 ;  The  vims  of  cattle  plague, 
370;  The  virus  of  rabies,  370;  Occurrence  and  nitration,  370;  Negri  bodies, 
370;  Rabies,  372;  Transmission,  372;  Diagnosis,  372;  Pasteur  treatment,  373; 
The  virus  of  hog  cholera,  373 ;  Spirochaeta  suis,  374;  Immunity,  374;  The  virus 
of  dengue  fever,  374;  The  virus  of  phlebotomus  fever,  374;  The  virus  of 
poliomyelitis,  374;  Occurrence  and  nitration,  374;  Resistance,  374;  Cultures, 
375;  Globose  bodies  of  Flexner  and  Noguchi,  375;  Transmission,  375;  The 
virus  of  measles,  375 ;  The  virus  of  typhus  fever,  375 ;  The  virus  of  small-pox, 
376;  Filtration,  376;  Small-pox,  376;  Vaccinia,  376;  Immunity,  376;  The 
virus  of  chicken  sarcoma,  377. 

CHAPTER  XXVII.     MASTIGOPHORA. 

Herpetomonas  muscae,  378;  Leptomonas  culicis,  378;  Cultures,  378;  Trypano- 
soma  rotatorium,  379;  Trypanosoma  lewisi,  381 ;  Tansmission,  382;  Cultures, 
383;  Pathogenesis,  383;  Immunity,  384;  Trypanosoma  brucei,  384;  Occurrence 
and  morphology,  384;  Transmission,  386;  Cultures,  386;  Nagana,  386;  Diag- 
nosis, 387;  Trapanosoma  evansi,  387;  Trypanosoma  equiperdum,  387;  Try- 
panosoma equinum,  388;  Trypanosoma  gambiense,  388;  Occurrence  and 
morphology,  388;  Transmission,  388;  Animal  inoculation,  389;  Human  try- 
panosomasis,  389;  Trypanosoma  rhodesiense,  390;  Trypanosoma  avium, 
391;  Occurrence,  391;  Cultures,  392;  Schizotrypanum  cruzi,  392;  Occurrence 
and  morphology,  392;  Animal  inoculation  394;  Cultures,  394;  Leishmania 
donovani,  394;  Occurrence  and  morphology,  394;  Cultures,  394;  Trans- 
mission, 396;  Kala-azar,  396;  Leishmania  tropica,  396;  Cultures,  397; 
Leishmania  infantum,  397 ;  Trypanoplasma  borreli,  398 ;  Bodo  lacertae,  398 ; 
Trichomonas  hominis,  400 ;  Lamblia  intestinalis,  400 ;  Mastigamceba  aspera, 
400;  Trimastigamceba  philippinensis,  400. 


XVI  TABLE  OF  CONTENTS 

CHAPTER  XXVIII.     RHIZOPODA. 

Amoeba  proteus,  401 ;  Occurrence  and  morphology,  401;  Cultures,  402;  Entamoeba 
coli,  402 ;  Occurrence  and  morphology,  402;  Parasitic  relation,  403 ;  Entamoeba 
tetragena,  404;  Occurrence  and  morphology,  404;  Parasitic  relation,  405; 
Entamoeba  histolytica,  405;  Relation  of  amebae  to  dysentery,  406;  Cultures 
of  dysenteric  amebae,  406;  Other  rhizopoda,  407. 

CHAPTER  XXIX.     SPOROZOA. 

Cyclospora  caryolytica,  408;  Occurrence  and  morphology,  408;  Pathogenesis,  410; 
Eimeria  steidae,  410;  Occurrence  and  morphology,  410;  Sexual  and  asexual 
cycles,  410;  Coccidiosis,  411;  Eimeria  schubergi,  412 ;  Haemoproteus  columbae, 
412;  Occurrence  and  morphology,  412;  Developmental  cycle,  412;  Haemopro- 
teus  danilewskyi,  414;  Fertilization  in  the  sexual  cycle,  414;  Haemoproteus 
ziemanni,  415;  Developmental  stages,  416;  Proteosoma  praecox,  417;  Occur- 
rence 418;  Cycle  in  the  blood,  418;  Sexual  cycle,  419;  Plasmodium  falciparum, 
419;  Morphology,  420;  Sexual  cycle,  422;  Cultures,  423;  Plasmodium,  vivax, 
424;  Cycle  in  the  blood,  424;  Sexual  cycle,  425;  Plasmodium  malariae,  425; 
Developmental  cycle,  425;  Malaria,  425;  Types  of  fever,  426;  Diagnosis,  427; 
Mosquito  carrier,  427;  Prevention,  427;  Plasmodium  kochi,  429;  Babesia 
bigemina,  429 ;  Morphology,  429;  Transmission,  429;  Texas  fever,  430;  Babesia 
canis,  430;  Gregarina  blattarum,  430 ;  Nosema  bombycis,  431 ;  Developmen- 
tal cycle,  431;  Pebrine,  432. 

CHAPTER  XXX.     CILIOPHORA. 

Paramaecium  caudatum,  433;  Morphology,  433;  Conjugation,  433;  Opalina  ran- 
arum,  434;  Balantidium  coli,  435;  Parasitic  relationships,  436;  Balantidium 
minutum,  437 ;  Sphaerophyra  pusilla,  437. 

INDEX  OF  NAMES 439 

INDEX  OF  SUBJECTS 445 


LIST  OF  ILLUSTRATIONS. 

FIG.  PAGE 

1.  Image  formation  by  means  of  a  pin-point  aperture 16 

2.  Image  formation  by  a  single  lens *7 

3.  Image  formation  by  two  lenses  in  series,  without  magnification 17 

4.  Image    formation  by  two  lenses  in   series,   with   magnification  of   two 

diameters l8 

5.  Image  formation  by  two  lenses  in  series,  with  magnification  of  three 

diameters l8 

6.  Microscope  objectives 20 

7.  Sectional  view  of  compound  microscope 21 

8.  Image  formation  in  the  compound  microscope 22 

9.  Image  formation  in  the  compound  microscope  with  an  eye-piece  of  higher 

power 22 

10.  Central  illumination  by  the  Abbe  condenser 24 

11.  Illumination  by  a  hollow  cone  of  light 24 

12.  Illumination  by  a  broad  convergent  beam 24 

13.  Dark-field  condenser 25 

14.  Optical  parts  of  dark-field  condenser 25 

15.  Production  of  the  "dark  outline  picture" 26 

16.  Production  of  the  "bright  outline  picture" 27 

17.  Obliteration  of  outline  by  homogeneous  illumination 27 

18.  Microscope 29 

19.  Abbe  condenser 3° 

20.  Platinum  needles 32 

21.  Pasteur  pipettes 33 

22.  Hanging-drop  preparation 34 

23.  Cornet  cover-glass  forceps 38 

24.  Stewart  cover-glass  forceps 38 

25.  Novy's  cover-glass  forceps 38 

26.  Kirkbride  forceps  for  slides 39 

27.  Schanze  microtome 57 

28.  Hot-air  sterilizer 65 

29.  Koch's  steam  sterilizer 66 

30.  Diagram  of  the  Arnold  steam  sterilizer 67 

31.  Steam  sterilizer,  Massachusetts  Board  of  Health 68 

32.  Autoclave 69 

33.  Apparatus  for  filling  test  tubes 89 

34.  Potato  tube 91 

35.  Kock's  serum  sterilizer 93 

xvii 


XV111  LIST  OF  ILLUSTRATIONS 

FIG.  PAGE 

36.  Pipette  for  collection  of  sterile  blood 95 

37.  Pipette  for  collection  of  sterile  blood  from  an  animal 96 

38.  Instrument  for  collection  of  feces  from  infants 102 

39.  Method  of  inoculating  culture  media 107 

40.  Petri  dish 108 

41.  Colonies  in  gelatin  plate 109 

42.  Manner  of  making  Esmarch  roll-tube no 

43.  Dilution  cultures  in  Esmarch  roll-tubes in 

44.  Stab-culture  closed  with  rubber  stopper 114 

45.  Smear  culture  closed  with  rubber  cap 114 

46.  Incubator 116 

47.  Reichert  gas-regulator 117 

48  MacNeal  gas-regulator 118 

49.  Pvoux  bimetallic  gas-regulator 119 

50.  Koch  automatic  gas-burner 119 

51.  Diagram  of  electric  regulator  for  low- temperature  incubator 122 

52.  Anaerobic  culture  by  Buchner's  method 126 

53.  Novy  anaerobe  jar  for  tube  cultures 127 

54.  Novy  anaerobe  jar  for  Petri  dishes  or  tubes 127 

55.  Novy  anaerobe  jar,  improved  pattern 127 

56.  Tripod  and  siphon  flask  for  anaerobic  culture  by  combined  hydrogen  and 

pyrogallate   method 128 

57.  Anaerobic  organism  that  will  not  grow  under  a  cover-glass 129 

58.  Method  of  making  collodion  capsules 135 

59.  Common  molds 138 

60.  Yeast  cells  stained  with  fuchsin 139 

61.  Wine  and  beer  yeasts 140 

62.  Various  groupings  of  micrococci 142 

63.  Bacilli  of  various  forms 145 

64.  Sporulation 145 

65.  Various  positions  of  spores 146 

66.  Types  of  spirilla 147 

67.  Bacteria  with  capsules 148 

68.  Bacteria  showing  flagella 148 

69.  Formation  of  spores 149 

70.  Bacteria  with  spores 149 

71.  Germination  of  spores 149 

72.  The  most  important  trypanosomes 152 

73.  Lcishmania  donovani 152 

74.  Leishmania  donovani  in  culture 153 

75.  Trichomonas  hominis 153 

76.  Lamblia  intestinalis 1 53 

77.  Entamosba  coli 154 

78.  Developmental  cycle  of  Eimeria  sckubergi ! .  . .  .  156 

79.  Asexual  cycle  of  Plasmodium  falciparum 15? 


LIST  OF  ILLUSTRATIONS  XIX 

FIG.  PACK 

80.  Forms  of  Babesia  muris 158 

8 1.  Developmental  cycle  of  Nosema  bombycis I5° 

82.  Sedgwick-Tucker  aerobioscope 177 

83.  Jeffer's  plate  for  counting  colonies 184 

84.  Surface  divided  in  square  centimeters  for  counting  colonies 185 

85.  Receptor  of  the  first  order  uniting  with  toxin 209 

86.  Receptor  of  the  second  order 210 

87.  Receptor  of  the  third  order 214 

88.  Deviation  of  complement 216 

89.  Mucor  mucedo 232 

90.  Mucor  corymbifer 232 

91.  Aspergillus  glaucus 233 

92.  Penicillium  crustaceum 234 

93.  Oidium  lactis 236 

94.  Oidium  albicans,  colony 237 

95.  Oidium  albicans,  mycelial  thread 238 

96.  Scutulum  of  favus  on  the  arm  of  a  man 239 

97.  Scutulum  of  favus  in  a  mouse 240 

98.  Achorion  schoenleinii,  colony 241 

99.  Sporotrichum  schenki,  cultures  on  agar 242 

100.  Sporotrichum  schenki,  forms  of  mycelium 243 

101.  Organisms  found  in  oidiomycosis 245 

102.  Actinomyces  bovis 247 

103.  Gonococci  and  pus  cells 251 

104.  Meningococcus  in  spinal  fluid 256 

105.  Pneumococcus  showing  capsule 258 

106.  Staphylococcus  aureus,  gelatin  culture 265 

107.  Bacillus  subtilis 269 

108.  Anthrax  bacilli  in  capillaries  of  the  liver 270 

109.  Bacterium  anthracis  showing  spores 271 

no.  Bacterium  anthracis,  colony  upon  a  gelatin  plate 271 

in.  Bacterium  anthracis,  thread  formation  of  colony 272 

112.  Bacillus  welchii,  in  agar  showing  gas  formation 278 

113.  Tetanus  bacilli,  showing  terminal  spores 280 

1 14.  Bacillus  tetani,  stab  culture 281 

115.  Bacillus  botulinus 282 

116.  Bacillus  of  diphtheria 286 

117.  Bacillus  diphtheria  stained  by  Neisser's  method 286 

118.  Forms  of  Bacillus  diphtheria  in  cultures  on  Loeffler's  serum 287 

119.  Forms  of  Bacillus  diphtheria  on  agar 287 

1 20.  Colonies  of  Bacillus  diphtheria  on  glycerin  agar 288 

121.  B.  diphtherias,  culture  on  glycerin  agar 289 

122.  Swab  and  culture  tube  for  diagnosis  of  diphtheria 290 

123.  The  Morax-Axenfeld  bacillus 296 

1 24.  The  Koch-Weeks  bacillus 297 


XX  LIST  OF  ILLUSTRATIONS 

FIG.  PAGE 

125.  Bacillus  tuberculosis  in  sputum 300 

126.  Bacillus  tuberculosis  from  a  pure  culture 301 

127.  Tubercle  bacillus  showing  branching  and  involution  forms 302 

128.  Bacillus  tuberculosis,  culture  on  glycerin  agar 303 

129.  Bacillus  of  bubonic  plague 317 

130.  Bacillus  coli,  showing  flagella 324 

131.  Bacillus  coli,  superficial  colony  on  gelatin  plate 325 

132.  Friedlander's  pneumobacillus,  gelatin  stab-culture 327 

133.  Bacillus  of  typhoid  fever 330 

134.  Bacillus  typhosus,  showing  flagella 331 

135.  Colonies  of  Bacillus  typhosus  and  Bacillus  coli 331 

136.  Bacillus  mallei 339 

137.  Cholera  vibrios,  short  form 346 

138.  Cholera  vibrios,  showing  flagella 347 

139.  Involution  forms  of  the  spirillum  of  cholera 347 

140.  Spirochaetae  of  relapsing  fever 354 

141.  SpirochcBta  recurrentis  in  blood  of  a  rat 355 

142.  Preparation  showing  Spirochata  pallida  and  Spirochceta  refringens 357 

143.  Spirochata  pallida  stained  by  Levaditi  method 359 

144.  Aedes  (Stegomyia)  calopus 369 

145.  Negri  bodies  in  brain  of  a  rabid  dog 371 

146.  Herpetomonas  musc<z 378 

147.  Leptomonas  culicis 379 

148.  Trypanosoma  rotatorium  in  blood  of  a  frog 380 

149.  Trypanosoma  rotatorium  in  culture 380 

150.  Trypanosoma  lewisi 381 

151.  Trypanosoma  lewisi,  various  multiplication  forms •. .  .  382 

152.  Trypanosoma  lewisi,  eight-cell  rosette 383 

153.  The  most  important  trypanosomes  parasitic  in  vertebrates 384 

154.  Glossina  morsitans,  dorsal  view 385 

155.  Glossina  morsitans,  lateral  view 385 

156.  Trypanosoma  equiperdum 387 

157.  Glossina  palpalis : 389 

158.  Trypanosoma  avium  in  blood  of  birds 39° 

159.  Trypanosoma  avium  in  culture 391 

1 60.  Schizotrypanum  cruzi  in  tissues  of  the  guinea  pig 393 

161.  Schizotrypanum  cruzi  in  human  blood 394 

162.  Conorhinus  megistus 395 

163.  Leishmania  donovani  in  spleen  juice 395 

164.  Leishmania  donoiani  in  culture 396 

165.  Leishmania  tropica  in  pus 396 

166.  Leishmania  tropica  in  culture 397 

167.  Trypanoplasma  cyprini 397 

168.  Bodo  lacerta 398 

169.  Trichomonas  hominis 398 


LIST  OF  ILLUSTRATIONS  XXI 

FIG.  PAGE 

170.  Lamblia  intestinalis 399 

171.  Trimastigamceba  philippinensis 399- 

172.  Amoeba  proteus 4°i 

173.  Entamceba  coll 4°2 

1 74.  Entamceba  tetragena,  unstained 4°4 

175.  Entamceba  tetragena,  stained  preparation 4°4 

176.  Entamceba  tetragena,  mature  cyst 4°S 

177.  Cydospora  caryolytica,  male  cells 408 

178.  Cydospora  caryolytica,  female  cells 4°9 

179.  Cydospora  caryolytica,  fertilization  and  production  of  sporozoits 409 

180.  Eimeria  steidce,  oocyst 410 

181.  Eimeria  steidce,  various  forms 411 

182.  Hcemoproteus  columbce,  developmental  cycle 4T3 

183.  Hcemoproteus  danilewskyi 414 

184.  Hcemoproteus  ziemanni,  gametocytes 415 

185.  Hcemoproteus  ziemanni,  formation  of  microgametes  and  fertilization 415 

186.  Hcemoproteus  ziemanni,  various  forms  observed  in  blood 4J6 

187.  Developmental  cycle  of  Protoesoma 417 

188.  Proteosoma  prcecox  in  blood  of  a  lark 418 

189.  Midgut  of  a  mosquito  showing  oocysts  of  Proteosoma 418 

190.  Oocyst  of  Proteosoma 4*9 

191.  Plasmodium  falciparum,  various  forms  observed  in  the  blood 420 

192.  Capillary  of  brain  filled  with  Plasmodium  falciparum 420 

193.  Plasmodium  falciparum,  development  of  the  gametocytes 421 

194.  Stomach  wall  of  Anopheles  infected  with  Plasmodium  falciparum 421 

195.  Digestive  tract  of  Anopheles,  infected  with  Plasmodium  falciparum 422 

196.  Plasmodium  falciparum,  ripe  sporozoits  in  oocyst 422 

197.  Salivaiy  gland  of  Anopheles,  containing  sporozoits  of  Plasmodium  falci- 

parum    423 

198.  Plasmodium  vivax,  stages  in  asexual  cycle 424 

199.  Plasmodium  vivax,  sporulation 424 

200.  Plasmodium  vivax,  double  infection 424 

201.  Plasmodium  vivax,  stages  in  development  of  the  gametocytes 425 

202.  Plasmodium  malarice,  asexual  cycle 426 

203.  Plasmodium  malaria,  gametocytes 426 

204.  Comparison  of  Culex  and  Anopheles 428 

205.  Babesia  bigemina 429 

206.  Gregarina  blattarum 431 

207.  Nosema  bombycis 432 

208.  Paramcecium  caudatum 433 

209.  Paramcecium  caudatum  and  Paramcecium  pittrinum 434 

210.  Opalina  ranarum 435 

211.  Balantidium  coll 436 

212.  Intestinal  wall  infected  with  Balantidium  coll 436 

213.  Sphcerophrya  pusilla  within  a  paramaecium 437 


INTRODUCTION. 


Bacteriology  and  Microbiology. — The  science  of  Bacteriology 
occupies  a  somewhat  peculiar  position  among  the  natural 
sciences,  partly  because  of  its  recent  development  and  partly 
because  of  the  overshadowing  importance  of  its  practical  appli- 
cations. As  bacteria  are  microscopic  plants,  some  have  con- 
sidered bacteriology  as  a  minor  division  of  botany;  but  the 
methods  of  work  and  the  practical  applications  of  bacteriology 
have  little  in  common  with  those  of  the  more  ancient  science. 
Indeed  were  it  not  for  the  importance  of  these  little  organisms 
to  the  chemist,  the  pathologist,  the  physician  and  the  agricul- 
turist, we  should  hear  little  about  them. 

The  foundations  of  the  science  were  laid  by  Pasteur  (1858) 
by  the  introduction  of  media  and  methods  for  artificial  culture 
of  bacteria  and  the  separation  of  mixtures  into  pure  culture  by 
the  laborious  and  uncertain  but  nevertheless  successful  method 
of  dilution  in  fluid  media,  thus  making  possible  the  accurate  ex- 
perimental study  of  microbes.  Robert  Koch  (1872-1882)  con- 
tributed much  to  the  establishment  of  the  new  science  by  intro- 
ducing the  use  of  solid  media  and  the  method  of  plating  for  the 
isolation  of  pure  cultures  and  especially  by  his  wonderful 
achievements  in  investigation  of  the  pathogenic  bacteria  by 
his  new  methods.  Koch  used  potatoes,  and  aqueous  humor 
and  blood  serum  rendered  solid  by  the  addition  of  gelatin.  He 
first  employed  the  anilin  dyes  in  staining  bacteria  (1877), 
microphotography  of  bacteria  (1877),  homogeneous  immersion 
objectives  and  the  Abbe  illuminating  apparatus  (1878).  Much 
of  our  modern  technic  has  been  devised  by  his  pupils  and 


2  INTRODUCTION 

colleagues.  The  commonly  used  meat-water-pepton-gelatin  was 
introduced  by  Loffler;  agar  by  Frau  Hesse. 

The  development  of  bacteriology  has  been  promoted  by  the 
work  of  biologists,  botanists,  chemists,  pathologists  and  agronom- 
ists, many  of  whom  have  been  willing  to  include  bacteriology 
as  a  subdivision  of  their  own  field.  The  practical  importance  of 
Bacteriology  to  these  various  fields  is  becoming  progressively 
more  evident.  The  relation  to  pathology  and  medicine  is  per- 
haps most  clearly  recognized,  although  the  importance  of  bac- 
teria in  chemical  technology  and  in  agriculture  is  no  longer 
questioned.  The  relationships  to  general  biology  have  not  been 
so  completely  developed  as  yet,  partly  because  these  have  seemed 
to  offer  less  promise  of  immediate  practical  application,  and  partly 
because  few  well-trained  zoologists  or  botanists  have  devoted 
serious  attention  to  bacteriology. 

As  a  matter  of  fact,  bacteriology  must  be  ranked  as  a  distinct 
science,  especially  because  of  its  peculiar  special  technic  and  be- 
cause of  the  peculiarly  critical  thought  necessary  in  the  inter- 
pretation of  bacteriological  observations  and  experiments.  The 
importance  of  these  can  be  fully  appreciated  only  after  actual 
experience  in  handling  microbes.  Here  is  a  science  in  which 
skepticism  is  a  necessary  safeguard,  a  skepticism  which  will  be- 
come convinced  only  when  overwhelming  evidence  compels  con- 
viction; and,  while  regarding  other  conclusions  with  interest  or 
even  with  enthusiasm,  still  carefully  reserves  final  judgment  as 
long  as  the  observed  phenomena  are  open  to  more  than  one 
interpretation. 

These  methods  of  thinking  and  of  working  have  been  applied 
to  organisms  other  than  the  bacteria,  on  the  one  hand  to  the 
unicellular  animals,  the  protozoa,  on  the  other  to  more  complex 
plant-forms  such  as  the  yeasts  and  molds,  and  more  especially  to 
the  study  of  the  still  undefined  types  of  living  things  known  as 
filterable  viruses  or  more  vulgarly  as  the  ultramicroscopic  mi- 
crobes. Inasmuch  as  many  of  these  live  as  parasites  and  some 
are  important  in  the  causation  of  disease,  they  are  commonly 


INTRODUCTION  3 

considered  along  with  the  pathogenic  bacteria.  The  terms  mi- 
crobe and  micro-organism  properly  include  these  as  well  as  the 
bacteria.  There  is  thus  an  evident  tendency  to  extend  the  field 
of  bacteriology  so  that  it  becomes  microbiology  or  the  science  of 
micro-organisms.  There  are  many  reasons  why  this  is  desir- 
able. It  is  certainly  essential  that  the  microbes  included  among 
the  protozoa  and  the  filterable  viruses  should  receive  more  atten- 
tion in  the  future,  both  from  beginning  students  and  from  trained 
investigators.  Until  separate  instruction  in  these  subjects  is  pro- 
vided for  medical  students,  they  may  perhaps  best  be  studied 
along  with  bacteriology. 

Biological  Relationships. — Since  the  earliest  times,  the  essen- 
tial difference  between  living  things  and  lifeless  things,  that  is, 
the  nature  of  life,  has  been  an  interesting  subject  of  speculation. 
It  was  at  first  assumed  as  a  matter  of  course  that  the  transition 
from  lifeless  to  living  matter  readily  took  place  without  the 
agency  of  preexisting  living  matter.  This  speculative  assump- 
tion is  still  not  without  its  able  supporters.  The  history  of  actual 
observations,  however,  is  one  long  record  of  refutation  of  this 
assumption  wherever  the  facts  have  been  subjected  to  accurate 
observation.  The  ancient  Greeks  held  that  living  beings  arose 
spontaneously  and  even  Aristotle  (384  B.C.)  asserted  that  ani- 
mals were  sometimes  formed  in  this  way.  These  ideas  were  dis- 
proved by  more  careful  observation.  A  notable  experiment  was 
that  of  Francesco  Redi  (about  1650)  who  allowed  meat  to  putrefy 
in  a  jar  covered  with  fine  wire  gauze.  The  flies  attracted  by  the 
odor  deposited  their  eggs  on  the  gauze  and  the  maggots  were 
hatched  there.  The  assumption  that  the  maggots  arose  de  novo 
in  putrefying  meat  was  thus  disproven.  Harvey  in  1650  made  the 
famous  statement,  "Omne  animal  ex  ovo"  which  was  later  ex- 
tended to  "Omne  vivum  ex  vivo." 

When  Anthony  van  Leeuwenhoek,  the  "Father  of  micro- 
scopy," discovered,  described  and  figured  bacteria  in  1683,  the 
assumption  of  spontaneous  generation  was  at  once  applied  to  this 
group  of  organisms  and,  although  rendered  exceedingly  doubtful 


4  INTRODUCTION 

by  the  experiments  of  Spallanzani  (1777)  and  of  Schulze  (1836), 
it  still  continued  to  be  accepted  by  many  scientific  men  until  it 
was  combated  by  Pasteur,  1860  to  1872.  After  the  accurate 
observations  of  Pasteur  upon  fermentation  and  putrefaction  and 
his  successful  defense  of  them  through  a  long  period  of  contro- 
versy, the  assumption  of  spontaneous  generation  as  applied  to 
bacteria  was  discredited  and  has  been  very  generally  given  up. 
Only  a  very  few  observers1  still  claim  the  existence  of  evidence 
in  support  of  its  application  here.  The  more  prominent  advo- 
cates2 of  the  assumption  of  spontaneous  generation  or  abio- 
genesis  seem  inclined  now  to  apply  it  to  some  group  of  living 
beings  still  beyond  the  limits  of  actual  observation. 

Closely  related  to  the  assumption  of  abiogenesis  has  been  the 
assumption  of  heterogenesis  among  the  bacteria,  the  notion  that 
various  kinds  of  microbes  could  readily  be  produced  from 
one  species.  Although  very  successfully  combated  by  Pasteur, 
this  idea  still  persisted  for  many  years  in  the  early  bacteriological 
literature,  the  observed  new  species  of  microbes  actually  resulting 
from  faulty  technic  by  which  new  germs  had  gained  entrance  to  a 
former  pure  culture.  These  observations  are  often  repeated 
unwittingly  by  beginners  in  bacteriology.  The  validity  of  bac- 
terial species  is  now  unquestioned.  On  the  other  hand,  the  vari- 
ability in  the  descendants  of  a  single  cell  through  a  greater  or 
less  range,  and  the  possibility  of  producing  morphologically  and 
physiologically  different  strains  of  the  same  species  by  appro- 
priate environmental  conditions  are  now  well  known,  resulting 
again  very  largely  from  the  fundamental  work  of  Pasteur  in  the 
production  of  attenuated  cultures  of  the  germs  of  chicken  cholera, 
and  of  anthrax. 

The  systematic  relationships  and  the  classification  of  bacteria 
were  first  studied  by  O.  F.  Mueller  (1786).  Ehrenberg  (1838) 
made  the  first  serious  attempt  at  a  comprehensive  classification 

1  Bastian,  The  Evolution  of  Life,  London,  1907.     The  Origin  of  Life,  London, 

1913- 

2  Schafer,  Nature,  Origin  and  Maintenance  of  Life,  Science,  1912,  Vol.  XXXVI, 
pp.  289-312. 


INTRODUCTION  5 

and  many  modern  systematists  are  inclined  to  return  to  his  work 
to  establish  authoritative  terminology  for  present  use.  He  re- 
garded the  bacteria  as  animals.  Ferdinand  Cohn  (1872)  recog- 
nized the  nature  of  bacterial  spores,  showed  the  close  relation- 
ship of  bacteria  to  the  algae  and  established  their  classification 
in  the  plant  kingdom.  He  distinguished  six  genera — micro- 
coccus,  bacterium,  bacillus,  vibrio,  spirillum  and  spirochaeta. 
Migula  (1897)  undertook  an  extensive  revision  of  bacteriological 
nomenclature  and  classification,  basing  it  upon  morphological 
characters,  and  his  system  is  doubtless  the  most  satisfactory  yet 
offered.  The  subject  is  still  in  a  very  unsettled  state,  neverthe- 
less, and  there  is  no  system  of  classification  generally  accepted 
by  bacteriologists.  The  problem  presents  so  many  difficulties 
and  our  knowledge  of  the  bacteria  is  still  so  incomplete  that  many 
authorities  seem  prone  to  consign  systematic  classification  to  the 
future,  and  to  employ  names  of  sufficient  historical  prominence 
to  insure  their  correct  interpretation. 

Fermentation  and  Putrefaction.  —  The  relation  of  micro- 
organisms to  the  decomposition  of  organic  matter,  fermentation 
and  putrefaction,  was  one  of  the  first  fields  of  applied  bacteri- 
ology to  be  studied.  Following  the  observation  of  bacteria  in 
saliva  by  van  Leeuwenhoek  in  1683,  micro-organisms  were  dis- 
covered in  all  sorts  of  decomposing  material.  At  first,  these  or- 
ganisms were  regarded  as  unimportant  for  the  chemical  process 
and  interest  attached  chiefly  to  the  question  of  their  origin, 
whether  by  spontaneous  generation  or  from  previously  living 
cells.  Needham  (1745)  directing  his  attention  more  particularly 
to  this  first  question,  boiled  an  infusion  of  meat,  and  keeping  it  free 
from  contact  with  the  air,  nevertheless  observed  after  some  days 
the  presence  of  "infusoria."  Spallanzani  (1765)  repeated  Need- 
ham's  experiments,  subjecting  hermetically  sealed  flasks  of  meat 
infusion  to  the  temperature  of  boiling  water  for  one  hour, 
and  he  found  no  subsequent  development  of  life  and  no  decom- 
position of  the  infusion  as  long  as  it  remained  sealed.  While 
discussion  continued  concerning  the  discrepancy  between  the 


6  INTRODUCTION 

results  of  Needham  and  Spallanzani  and  concerning  the  relation 
which  the  subsequent  exclusion  of  the  air  might  bear  to  the  ab- 
sence of  life  in  the  flasks,  .the  method  of  heating  was  applied  to 
the  preservation  of  vinegar  by  Scheele  (1782)  and  to  the  preser- 
vation of  foods  in  general  by  Appert  (1811).  The  method  was 
quickly  introduced  into  other  countries,  and  developed  by  va- 
rious tradesmen,  who  attempted  with  more  or  less  success  to 
keep  their  processes  secret.  Success  in  preservation  by  canning 
remained  somewhat  uncertain,  as  a  precise  understanding  of  the 
underlying  scientific  principles  was  still  lacking.  Schulze  (1836) 
showed  that  air  might  be  admitted  to  flasks  prepared  by  Spallan- 
zani's  method,  without  the  development  of  life  and  without 
putrefaction,  provided  the  air  were  first  passed  through  a  series 
of  bulbs  containing  concentrated  sulphuric  acid.  The  subse- 
quent work  of  Schroder  and  van  Dusch  (1853),  who  obtained 
similar  success  by  filtering  the  air  through  cotton,  of  Pasteur  and 
Tyndall  (1860-62)  who  were  able  to  preserve  putrescible  fluids 
directly  in  contact  with  air,  provided  the  air  were  rendered  per- 
fectly free  from  dust,  has  established  the  fact  that  the  decom- 
position ordinarily  taking  place  after  exposure  to  the  air  is  due 
to  the  introduction  of  living  germs  into  the  previously  sterile 
material. 

The  idea  that  specific  kinds  of  fermentation  are  caused  by 
specific  kinds  of  microbes  was  first  clearly  put  forward  by  Schwann 
and  Cagniard-Latour  (1837),  who  showed  that  yeast-cells  were 
living  organisms  and  claimed  that  the  alcoholic  fermentation  of 
sugar  solutions  was  due  to  their  growth.  The  importance  of  this 
relationship  received  little  recognition  until  Pasteur  (1860-72), 
during  his  extensive  and  careful  researches  into  the  nature  of 
fermentation  and  the  causation  of  undesirable  fermentation  (dis- 
eases of  wines  and  beers),  demonstrated  conclusively  that  the 
kind  of  decomposition  of  a  fermentable  substance  depended  upon 
the  nature  of  the  substance,  the  kind  of  microbes  present  and  the 
environmental  conditions,  such  as  temperature  and  presence  or 
exclusion  of  air.  The  mere  introduction  of  a  small  number  of 


INTRODUCTION  7 

unfavorable  microbes  was  sufficient  to  change  the  whole  nature 
and  course  of  the  fermentation.  Furthermore,  Van  der  Brock 
(1857)  and  Pasteur  (1863)  were  able  to  collect  such  fermentable^ 
materials  as  grape  juice,  wine,  blood,  tissues  of  plants  and  ani- 
mals and  preserve  them  free  from  decomposition  and  from  all 
microbic  life,  merely  by  effectively  avoiding  contact  with  germs 
during  collection  and  storage. 

The  agency  of  microbes  in  fermentation  was  ridiculed  by 
Liebig,  the  most  prominent  chemist  of  the  time,  who  steadfastly 
continued  to  regard  decomposition  of  organic  material  as  a 
purely  chemical  process  uninfluenced  by  biological  activity.  His 
ideas  prevailed  for  a  time  because  of  his  prominent  position. 
The  correctness  of  Pasteur's  contention  is  now  universally  ac- 
cepted. Nevertheless  it  should  not  be  forgotten  that  many 
organic  substances  are  in  themselves  so  unstable  that  even  in  the 
absence  of  microbic  life  they  disintegrate,  or  become  oxidized  in 
the  presence  of  the  air.  These  changes  are  different  from  those 
ordinarily  known  as  fermentation  and  putrefaction. 

Pathology  and  Hygiene. — The  history  of  the  development  of 
our  ideas  concerning  the  relation  between  microbes  and  disease 
is  one  of  the  most  interesting  and  perhaps  the  most  important 
chapter  in  the  history  of  bacteriology.  The  customs  and  rec- 
ords of  the  ancients  give  evidence  that  they  recognized  the  pres- 
ence of  an  unseen  agency  in  the  body  of  the  diseased  individual 
capable  of  causing  sickness  in  others.  This  was  recognized  by 
the  ancient  Persians  as  recorded  by  Herodotus.  The  isolation 
of  lepers  by  the  ancient  Hebrews  shows  that  the  infectious  char- 
acter of  the  disease  has  long  been  recognized,  though  other  affec- 
tions than  leprosy  were  probably  confused  with  this  disease.  "He 
is  unclean;  he  shall  dwell  alone;  without  the  camp  shall  his 
habitation  be."  (Lev.  XIII,  46).  There  is,  in  fact,  much  in  the 
laws  of  Moses  that  points  to  some  knowledge  of  the  nature  of 
infection.  "This  is  the  law,  when  a  man  dieth  in  a  tent  all  that 
come  into  the  tent  and  all  that  is  in  the  tent  shall  be  unclean  for 
seven  days.  And  every  open  vessel  that  has  no  covering  on  it 


8  INTRODUCTION 

shall  be  unclean."  (Numb.  XIX,  14,  15).  "  Every  thing  that  may 
abide  the  fire,  ye  shall  make  it  go  through  the  fire,  and  it  shall  be 
clean."  (Numb.  XXXI,  23).  In  Homer  we  read  of  Ulysses,  that, 
having  slain  his  wife's  troublesome  suitors: 

"With  fire  and  sulphur,  cure  of  noxious  fumes, 
He  purged  the  walls  and  blood-polluted  rooms."  (Pope's  Odyssey). 

These  records  certainly  suggest  a  rather  advanced  state  of  knowl- 
edge concerning  the  nature  of  contagion.  It  may  be  that  they 
record  customs  derived  from  a  superior  knowledge  of  some  other 
ancient  people,  perhaps  the  ancient  Egyptians.  During  the 
middle  ages,  as  doubtless  also  before  the  dawn  of  history,  epi- 
demic disease  was  regarded  as  a  visitation  of  Providence  or  at- 
tributed to  the  influence  of  gods,  demons  or  other  supernatural 
agencies.  Epidemics  were  associated  with  the  appearance  of 
comets  in  the  sky  or  with  other  evidences  of  divine  wrath.  These 
conceptions  of  disease  have  not  altogether  disappeared  even  at 
the  present  time. 

Hippocrates  (400  B.  C.)  denied  the  supernatural  causation 
of  disease  and  held  that  such  doctrines  were  mere  cloaks  for  help- 
less ignorance.  He  ascribed  epidemic  disease  to  a  morbid  secre- 
tion of  the  atmosphere,  and  later  writers  have  expressed  this 
idea  of  a  morbid  secretion  by  the  word  miasm,  its  exact  nature 
remaining  for  centuries  intangible  and  mysterious.  There  is 
here  a  conception  different  from  that  upon  which  the  hygienic 
measures  of  the  Persians  and  Hebrews  were  founded  and  the 
distinction  was  clearly  expressed  by  Pettenkofer  in  the  nineteenth 
century,  who  defined  contagious  diseases  as  those  which  are  trans- 
mitted directly  from  man  to  man  or  through  the  agency  of  solid 
objects,  while  in  miasmatic  diseases  the  causative  agent  enters 
from  the  outside  world  where  it  may  live  naturally  or  where  it 
must  have  undergone  a  ripening  process  since  its  escape  from  the 
body  of  the  sick  person.  As  will  be  seen  later  these  ideas  apply 
very  well  to  certain  diseases,  for  example,  small-pox  and  syphilis 
as  contagious  diseases  and  yellow-fever  and  malaria  as  a  mias- 


INTRODUCTION  9 

matic.  The  ancient  Greeks  recognized  the  contagiousness  of 
several  diseases  and  Galen  classed  plague,  itch,  ophthalmia,  con- 
sumption and  rabies  as  contagious.  Fracas torius  (1546)  during 
the  period  of  the  great  epidemic  of  syphilis  in  Europe,  published 
a  book  containing  the  first  comprehensive  discussion  of  the  theory 
of  contagion.  He  recognized  contagion  by  contact,  by  fomites 
and  at  a  distance.  Soiled  material  of  all  kinds  was  included  un- 
der fomites,  as  also  those  healthy  individuals  capable  of  trans- 
mitting disease,  a  phenomenon  already  recognized.  Transmis- 
sion by  insects  and  animals  was  also  included  under  this  head. 
The  transmission  "per  distans"  was  considered  due  to  emana- 
tions from  the  patient  diffusing  to  a  distance  through  the 
atmosphere. 

Kircher  in  1658  claimed  to  have  seen  the  living  contagium  in 
the  body  in  the  form  of  minute  worms,  and  his  observations  were 
widely  recognized.  The  objects  he  saw  were  not  accurately 
described  but  it  seems  very  certain  that  they  were  not  bacteria. 
Probably  they  were  the  normal  cells  of  the  tissues. 

The  discovery  of  bacteria  by  van  Leeuwenhoek  (1683)  was 
not  immediately  recognized  as  of  importance  for  the  germ 
theory.  Leeuwenhoek  himself  considered  it  impossible  for  his 
"animalcula"  to  penetrate  into  the  blood  because  of  the  com- 
pactness of  the  epithelial  tissues. 

Almost  a  century  later,  Plenciz  (1762)  maintained  that  each 
infectious  disease  must  have  its  own  specific  cause.  Reimarus 
(1794)  also  expressed  the  same  opinion  and  considered  these  liv- 
ing organisms  to  be  of  the  order  of  infusoria  or  perhaps  still 
smaller  beings  not  yet  visible  with  the  microscope.  These  ideas 
were  not  supported  by  objective  evidence  and  received  only 
passing  attention.  They  were  soon  thrust  aside  by  other  inter- 
esting if  less  valuable  speculations. 

The  development  of  general  knowledge  of  the  animalcules 
in  the  early  part  of  the  nineteenth  century,  already  referred  to 
in  the  discussion  of  the  biological  relationships  and  of  fermentation, 
was  preparing  the  way  for  progress  in  the  problem  of  disease. 


10  INTRODUCTION 

In  1834  the  contaguim  vivum  of  itch,  the  itch  mite  (Sar copies 
scabei),  a  fairly  large  mite  to  be  sure,  was  rediscovered  and  its 
relation  to  the  disease  made  evident.  In  1837,  the  same  year 
in  which  Cagniard-Latour  and  Schwann  established  the  relation 
of  living  yeast  to  alcoholic  fermentation,  Donne  described 
vibriones  (bacteria)  in  syphilitic  ulcers,  and  Audouin  amplified 
the  discovery  of  Bassis  that  muscardine,  a  disease  of  the  silk- 
worm, was  caused  by  a  mold  (Botrytis  bassiana)  which  was  trans- 
mitted from  the  sick  to  the  healthy  worms  by  contact  or  by  air 
currents.  These  discoveries  furnished  a  great  impulse  to  further 
investigation. 

Henle  (1840)  reviewed  the  evidence  then  at  hand  and  con- 
cluded in  a  very  logical  way  that  the  causes  of  contagious  dis- 
eases were  to  be  sought  for  among  the  minute  living  micro-organ- 
isms. He  recognized  that  no  human  disease  had  yet  been  shown 
to  be  caused  by  a  micro-organism  and  he  formulated  the  re- 
quirements to  be  fulfilled  in  order  to  prove  such  a  relation, 
namely,  that  the  microbe  must  be  constantly  present  in  the 
disease,  must  be  isolated  from  the  infectious  material,  and  must 
then  alone  be  capable  of  producing  the  disease. 

During  the  next  twenty  years,  the  attempts  to  discover  the 
cause  of  an  infectious  disease  and  to  satisfy  the  postulates  of  Henle 
were  successful  in  several  diseases  due  to  molds,  Favus  (Achorion 
Schoenleinii)  1839,  similar  skin  diseases  known  as  trichophytosis 
and  pityriasis  and  especially  thrush,  shown  to  be  caused  by 
Oidium  albicans  by  Robin  in  1847;  but  in  all  the  more  important 
diseases  only  failure  resulted.  The  reawakened  interest  in  con- 
tagium  vivum  therefore  again  gradually  faded  away. 

During  this  time  Pollender  and  Davaine  and  Rayer  (1850) 
had  discovered  the  minute  rods  in  the  blood  of  animals  sick  with 
anthrax,  and  in  1863  Davaine  had  proved  the  almost  constant 
presence  of  these  rods  in  the  disease  and  the  possibility  of  trans- 
mission by  inoculation  from  one  animal  to  another. 

Pasteur  from  1865  to  1868  investigated  the  fatal  disease  of 
silk-worms  known  as  pebrine,  discovered  the  microsporidium 


INTRODUCTION  1 1 

(Nosema  bombycis)  which  occurs  in  the  sick  worms  and  in  the 
eggs,  and  devised  a  successful  method  of  eradicating  the  disease.^ 

In  1870-71  the  presence  of  bacteria  in  wounds  and  in  the 
internal  purulent  collections  in  pyemia  and  septicemia  was  first 
definitely  recognized  by  Rindfleisch  (1870),  but  more  especially 
by  Klebs  in  a  large  number  of  cases  at  the  military  hospital  at 
Karlsruhe.  The  latter  observed  spherical  bacteria  arranged  in 
groups  or  as  a  rosary  to  which  he  gave  the  name  Microsporon 
septicum.  His  observations  were  quickly  confirmed  by  other 
competent  pathologists.  Similar  organisms  were  quickly  found 
in  a  great  many  wounds  and  other  inflammatory  processes. 
Specific  causal  relationship  was  still  unproven. 

In  1873  Obermeier  described  the  slender  but  actively  motile 
spirochetes  seen  by  him  in  the  blood  in  relapsing  fever  as  early 
as  1868. 

In  1874  Billroth  concluded  that  there  was  still  no  disease  in 
which  the  causal  relationship  of  micro-organisms  had  been  con- 
clusively proven.  The  skin  diseases  due  to  molds  were  relatively 
unimportant  and  had  not  been  recently  studied.  The  microbes 
found  in  other  diseases  might  just  as  reasonably  be  regarded  as 
a  product  of  the  disease  or  as  only  incidental  to  it.  Even  in 
anthrax,  where  the  evidence  seemed  strongest,  there  were  cases 
of  the  disease  without  the  presence  of  the  peculiar  rod-like  bodies 
in  the  blood,  and  indeed  these  rods  might  be  crystals  and  not 
living  organisms  at  all. 

Since  1867  Lister,  stimulated  by  the  investigations  of  Pasteur 
on  fermentation  and  putrefaction,  had  been  developing  and 
applying  an  antiseptic  method  to  the  treatment  of  wounds, 
which  consisted  of  the  use  of  carbolic  acid.  The  results  of  this 
method  published  in  1875  were  so  remarkably  favorable  that  it 
was  quickly  adopted  throughout  the  world,  and  its  success  did 
much  to  prepare  the  way  for  the  recognition  of  the  role  of  microbes 
in  suppuration,  if  it  did  not  in  itself  convince. 

Robert  Koch,  1876-1881,  first  satisfied  the  postulates  laid 
down  by  Henle,  and  again  formulated  by  himself,  in  the  bacterial 


12  INTRODUCTION 

disease,  Anthrax.  The  presence  of  the  bacilli  in  the  blood  of 
animals  suffering  from  anthrax  had  been  established  by  a  large 
number  of  previous  workers,  and  the  transmissibility  of  the  dis- 
ease by  inoculation  with  blood  of  diseased  animals  was  already 
known.  Koch  was  able  to  grow  the  bacillus  in  pure  culture  in  a 
test  tube,  using  the  aqueous  humor  of  the  ox's  eye  as  a  medium. 
He  was  able  to  observe  growth  and  division  and  the  formation 
and  germination  of  spores  under  the  microscope.  Finally  with 
these  cultures  which  had  been  propagated  a  long  time  in  the 
culture  medium,  he  was  able  again  to  cause  anthrax  by  injecting 
them  into  susceptible  animals.  The  demonstration  of  the  causa- 
tion of  disease  by  bacteria  had  been  achieved. 

The  introduction  by  Koch  in  1881  of  the  plate  method  of  sepa- 
rating bacteria  paved  the  way  for  rapid  advances  in  bacteriology, 
and  during  the  next  ten  years  the  bacterial  causes  of  several 
diseases  were  discovered  and  proven  by  thorough  test,  and  since 
then  the  number  of  diseases  known  to  be  due  to  bacteria  has 
gradually  increased. 

The  history  of  immunity  extends  far  back  into  ancient  times. 
For  many  diseases  it  was  recognized  that  those  who  recovered 
could  associate  with  the  sick  without  danger  to  themselves. 
Recognizing  this,  people  sometimes  exposed  themselves  purposely 
in  order  to  have  the  disease  at  a  convenient  time.  Artificial 
inoculation  to  cause  small-pox  was  introduced  into  Europe  from 
the  Orient  in  1721.  The  use -of  cowpox,  vaccination,  was  discov- 
ered by  Jenner  in  1797.  Artificial  immunization  by  inoculation 
with  altered  bacterial  cultures  was  first  successfully  demonstrated 
by  Pasteur  in  chicken  cholera  and  in  anthrax  in  1881.  Analogous 
methods  have  since  been  devised  for  many  other  diseases.  The 
discovery  of  the  antitoxic  property  of  the  blood  serum  of  animals 
immunized  to  tetanus  and  to  diphtheria  was  made  by  von  Behring 
and  Kitasato  (1891). 

With  the  discovery  of  amebae  in  the  stools  in  tropical  dysentery 
by  Loesch  (1875)  and  of  the  malarial  plasmodium  in  the  blood 
by  Laveran  (1880)  the  relationship  of  protozoa  to  important 


INTRODUCTION  13 

diseases  was  suggested.  An  enormous  number  of  protozoal  para- 
sites are  now  known,  many  of  them  associated  with  important 
diseases.  The  strict  proof  of  causal  relationship  to  the  disease 
has  presented  greater  difficulties  here,  especially  the  step  of  artifi- 
cial culture.  However,  the  causal  relationship  of  bacteria  hav- 
ing been  demonstrated,  the  probable  causal  relationship  of 
the  protozoa  has  found  more  ready  acceptance.  Cultures  of 
ameba  have  been  obtained  by  many  workers  but  the  successful 
cultivation  of  a  pathogenic  ameba  is  still  questionable.  Pure 
cultures  of  trypanosomes  were  obtained  by  Novy  and  his  pupils 
(1903-04)  and  the  infections  again  produced  by  inoculation  with 
these  cultures. 

The  transmission  of  protozoal  diseases  by  insects,  first  demon- 
strated by  Salmon  and  Smith  in  Texas  fever,  has  developed  into 
a  subject  of  prime  importance.  Malaria  and  the  insect,  Anophe- 
les, sleeping  sickness  and  tsetse  fly,  Glossina,  are  important  ex- 
amples of  this  relationship. 

Obermeier  (1873)  described  a  motile  spiral  organism  in  the 
blood  of  relapsing  fever,  the  first  known  parasitic  member  of  a 
group  of  very  great  importance.  Very  many  pathogenic  spiral 
organisms  of  this  general  type  are  now  known.  Their  syste- 
matic relationships  have  not  been  fully  worked  out  and  further 
knowledge  is  necessary  before  they  can  be  finally  classed  with 
either  the  bacteria  or  the  protozoa.  The  discovery  of  practical 
methods  of  artificial  culture  for  these  .organisms  has  been  very 
recent  and  the  most  successful  methods  seem  to  have  been  de- 
vised by  Noguchi  (1910-12).'  Many  of  these  parasites  are  trans- 
mitted by  insects  and  they  pass  through  a  somewhat  obscure  de- 
velopment in  the  insect  carriers,  the  forms  developed  being  ex- 
tremely minute  (Nuttall,  1912).  These  facts  suggest  a  possible 
relationship  of  this  group  of  organisms  to  the  filterable  viruses. 

Nocard  (1899)  discovered  that  the  virus  of  pleuro-pneumonia 
of  cattle  would  pass  through  filters  impervious  to  bacteria.  The 
number  of  recognized  filterable  viruses  has  grown  appreciably 
since  then  and  among  them  are  the  causes  of  several  very  im- 


14  INTRODUCTION 

portant  diseases,  such  as  yellow-fever,  dengue  fever,  poliomy- 
elitis, measles,  typhus  fever,  small-pox,  rabies  and  hog  cholera. 
Knowledge  of  this  group  of  organisms  is  accumulating  rapidly 
and,  although  microscopic  methods  of  denning  their  form  and 
structure  are  still  undeveloped,  they  cannot  with  justice  be  re- 
garded as  wholly  in  the  realm  of  the  unknown. 

Agriculture. — The  importance  of  microbes  in  soil  fertility 
and  agriculture  has  a  relatively  short  history.  Duclaux,  1885, 
showed  that  plants  could  not  well  utilize  complex  organic  matter 
as  food  in  the  absence  of  microbic  life.  In  addition  to  ordinary 
decomposition  of  organic  matter,  bacteria  also  bear  an  important 
relation  to  the  nitrogen  metabolism  of  plants.  Hellriegel  and 
Wilfarth  (1886-88)  showed  the  infectious  nature  of  the  nitro- 
gen-fixing root  tubercles  of  legumes,  and  the  organism  B.  radici- 
cola  was  isolated  by  Beyerinck  in  1888.  The  importance  for  agri- 
culture of  other  living  elements  in  the  soil,  such  as  amebae  and 
nematodes,  has  been  more  recently  recognized. 

Although  it  is  well  to  recognize  the  many  important  appli- 
cations of  bacteriology,  a  word  of  caution  may  not  be  amiss,  lest 
we  follow  too  eagerly  the  alluring  applications  and  neglect  the 
secure  foundation  of  scientific  knowledge  of  the  biology  and  bio- 
logical relationships  of  micro-organisms,  the  proper  training  in 
logical  thinking  concerning  these  beings  and  in  the  technic  of 
dealing  with  them. 


PART  I. 
BACTERIOLOGICAL  TECHNIC 


CHAPTER  I. 
THE  MICROSCOPE  AND  MICROSCOPIC  METHODS. 

The  development  of  bacteriology  has  depended  especially 
upon  the  development  of  new  methods  of  scientific  study,  and 
in  a  very  important  way  upon  the  improvements  in  construction 
of  the  microscope  and  in  methods  of  preparing  objects  for  study 
under  the  microscope.  Knowledge  of  the  construction  of  a  mi- 
croscope is  not  an  essential  part  of  bacteriology  but  the  demands 
of  modern  microscopical  methods  require  a  skill  in  manipulation 
of  the  instrument  which  is  best  acquired  after  the  principal  struc- 
tural features  of  the  microscope  are  understood. 

The  Development  of  the  Microscope. — Roger  Bacon,  in 
1276,  seems  to  have  been  the  first  to  recognize  the  peculiar  prop- 
erties of  a  lens.  Spectacles  began  to  be  used  about  the  same 
time  and  are  said  to  have  been  invented  by  d'Armato.1  Gali- 
leo (1610)  probably  made  the  first  record  of  the  use  of  the  com- 
pound microscope.  It  was  a  lens  maker,  Anton  van  Leeuwen- 
hoek,  who  first  saw  bacteria  in  1683.  A  method  of  correcting 
chromatic  aberration  was  discovered  by  Marzoli  in  1811,  but 
became  generally  known  through  the  work  of  Chevalier  in  1825. 
The  correction  of  the  color  defects  was  accomplished  by  the  com- 
bination of  two  kinds  of  glass,  crown  glass  and  flint  glass,  in  the 
1  Jour.  A.  M.  A.,  Nov.  9,  1912,  Vol.  LIX,  p.  1721. 


i6 


BACTERIOLOGY 


objective  lens  system,  and  made  possible  the  construction  of 
achromatic  objectives,  perhaps  the  most  important  advance  ever 
made  in  the  construction  of  the  microscope.  Abbe  (about  1880) 
introduced  his  substage  condenser  which  made  possible  the  in- 
tense illumination  of  the  microscopic  field.  In  collaboration  with 
Zeiss,  Abbe  (1886)  devised  an  objective  lens  system  with  more 
perfect  chromatic  correction  than  had  been  previously  attained. 
These  objectives  are  constructed  of  several  different  kinds  of 
glass  and  have  in  addition  one  lens  composed  of  rmorite.  Sieden- 
topf  and  Zsigmondi  (1903)  devised  a  method  of  illuminating  the 
microscopic  preparation  by  horizontal  beams  and  so  brought  to 


FIG.  i. — The  formation  of  an  image  by  means  of  a  simple  pin-point  aperture. 

(After  A,  E.  Wright.} 

view  exceedingly  minute  refractive  particles  as  luminous  points 
on  a  dark  field.  The  various  dark-field  condensers  introduced 
in  recent  years  (1906)  utilize  similar  principles,  the  object  being 
illuminated  by  oblique  light.  Recently,  Gordon  has  devised 
the  tandem  microscope,  an  instrument  which  has  demonstrated 
the  possibility  of  achieving  greater  microscopic  resolution  than 
has  previously  been  attained  and  even  suggests  that  there  is  no 
necessarily  final  limit  to  the  degree  of  magnification  at  which 
satisfactory  definition  and  resolution  may  be  achieved. 

Principle  of  the  Microscope. — The  formation  of  an  image  by 
means  of  a  simple  pin-point  aperture  is  illustrated  in  Fig.  i.  It 
will  be  noted  that  the  magnification  achieved  is  the  quotient  of 


THE   MICROSCOPE   AND   MICROSCOPIC   METHODS  17 

aperture-image  distance  divided  by  object-aperture  distance;  also 
that  the  sharpness  of  outline  of  the  image  increases  and  the 
brilliancy  diminishes  as  the  size  of  the  aperture  is  decreased. 

If  the  simple  aperture  be  replaced  by  a  convex  lens  and  the 
object  and  the  screen  be  set  at  the  conjugate  foci  of  the  lens,  it 


FIG.  2. — Image  formation  by  a  single  lens.  Note  that  the  image,  at  the  right, 
is  |  the  size  of  the  object,  in  proportion  to  their  respective  distances  from  the  lens; 
the  opening  angle  being  f  the  size  of  the  closing  angle. 

will  be  seen  that  magnification  is  again  the  quotient  of  the  aper- 
ture-image distance  divided  by  the  object-aperture  distance. 
The  sharpness  of  outline,  however,  depends  now  upon  the  quality 
of  the  lens  and  the  accurate  adjustment  of  the  distance,  and 
brilliancy  is  not  seriously  impaired  in  attaining  definition. 


FIG.  3. — Image  formation  by  two  lenses  in  series  without  magnification.  Note 
that  the  opening  angle  of  the  beam  preceding  from  the  object,  at  the  left,  is  equal  to 
the  closing  angle  of  the  beam  forming  the  image  at  the  right. 

Image  formation  in  the  human  eye  is  an  example  of  the  work- 
ing of  the  lens-armed  aperture.  The  rays  of  light  are  brought 
to  a  focus  on  the  retina  and  the  image  produced  here  is  in- 
verted and  actually  much  smaller  than  the  object,  the  reduction 
(minification)  being  again  measured  by  the  quotient  of  the  lens- 


i8 


BACTERIOLOGY 


retina  distance  divided  by  the  object-lens  distance.  The  longer 
the  antero-posterior  diameter  of  the  eye,  the  larger  will  be  the 
retinal  image.  Our  subjective  interpretation  of  the  stimulation 
of  the  retina  (i.e.,  what  we  see)  is  influenced  by  other  psycholog- 


FIG.  4. — Image  formation  by  two  lenses  in  series,  with  magnification  of  two 
diameters.  Note  that  the  opening  angle  of  the  beam  is  twice  as  large  as  the  closing 
angle. 

ical  elements  and  especially  by  the  memory  of  things  seen  before. 
When  two  lenses  are  disposed  in  series  so  that  the  rays  of 
light  coming  from  a  point  in  the  object  pass  through  both  lenses 
before  coming  to  a  focus,  we  find  the  possibilities  shown  in  Figs.  3, 
4  and  5.  In  the  figures  it  will  be  seen  that  the  image  produced 


FIG.  5. — Image  formation  by  two  lenses  in  series,  with  magnification  of  three 
diameters.  Note  that  the  opening  angle  of  the  beam  is  three  times  as  large  as  the 
closing  angle. 

when  the  first  lens  is  in  position  so  as  to  render  the  rays  parallel 
(Fig.  4),  is  just  five  times  as  large  as  that  produced  when  it  is 
left  out  (Fig.  2),  assuming  that  the  second  lens  is  capable  of 
change  so  as  to  focus  upon  the  same  screen  slightly  divergent 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        IQ 

rays  proceeding  from  the  object.  It  will  further  be  perceived 
that  the  sine  of  the  angle  of  divergence  of  the  beam  proceeding, 
from  the  object  varies  directly  with  the  magnification  achieved, 
and  further  that  the  magnification  in  any  such  system  is  equal 
to  the  quotient  of  the  sine1  of  the  angle  of  divergence  of  the  beam 
proceeding  from  the  object,  divided  by  the  sine  of  the  angle  of 
convergence  of  the  beam  to  form  the  image.  This  is  capable 
of  mathematical  proof  and  is  illustrated  in  the  four  figures. 
From  these  it  is  evident  that  magnification  is  a  function  of  the 
relation  of  these  two  angles  of  the  opening  and  closing  limbs  of 
the  beam,  and  that  the  intermediate  course  of  the  rays,  whether 
parallel,  convergent  or  divergent,  is  negligible  in  this  computation. 
If  the  second  lens  be  that  of  the  eye  and  an  image  is  to  be  formed 
on  the  retina,  then  the  rays  proceeding  from  a  point  must  be  ren- 
dered parallel,  or  approximately  so,  by  the  first  lens.  This  is  the 
arrangement  which  exists  in  the  simple  microscope  or  in  the  ordi- 
nary reading  glass.  The  magnification  achieved  by  such  a  simple 
microscope  is  measured  by  the  relation  between  the  magnitude 
of  the  image  on  the  retina  when  the  lens  is  employed,  and  the 
size  of  such  an  image  when  the  lens  is  left  out  of  the  path  of  the 
light.  The  value  of  the  reading  glass,  entirely  aside  from  con- 
siderations of  magnification,  in  conditions  of  hyperopia  and  pres- 
byopia is  also  evident  from  these  figures,  as  it  of  course  renders 
the  rays  coming  from  a  near  point  more  nearly  parallel,  and  thus 
enables  the  refracting  media  of  the  presbyopic  eye  to  bring  them 
to  a  focus. 

So  far  we  have  been  employing  in  our  discussion  the  ideal 
lens,  one  which  refracts  all  light  equally  and  brings  to  a  focus  in 
one  plane  all  rays  proceeding  from  one  plane  in  the  object.  As 
a  matter  of  fact  the  ideal  lens  in  this  sense  does  not  exist.  The 
simple  convex  lens  has  many  serious  optical  defects. 

1  In  the  figures,  as  drawn,  this  statement  actually  applies  to  the  tangents  of  the 
angles  designated,  rather  than  the  sines.  However,  for  very  small  angles  the  sine 
and  tangent  are  approximately  equal.  The  use  of  the  term  sine  finds  its  complete 
justification  in  the  fact  that  the  plane  at  which  the  rays  are  bent  is  not  flat  but  is 
the  segment  of  a  sphere  or  its  optical  equivalent. 


2O 


BACTERIOLOGY 


Points  in  the  same  plane  in  the  object  are  imaged  by  the  simple 
lens  on  a  curved  surface,  the  segment  of  a  spherical  surface. 
This  defect  is  known  as  spherical  aberration.  It  is  diminished 
to  some  extent  by  combining  convex  and  concave  lenses  and  the 
correction  may  be  changed  by  altering  the  distance  between  these 
component  lenses,  as,  for  example,  in  an  objective  equipped  with 
a  correction  collar.  Objectives  corrected  in  respect  to  spherical 
aberration  are  designated  as  aplanatic.  Restriction  of  the  size 
of  the  field  is  also  an  important  factor  in  making  it  appear  flat. 

Light  of  different  wave  lengths  (different  colors)  is  refracted 
to  a  different  degree  by  the  simple  lens,  so  that,  for  example,  the 
violet  rays  are  brought  to  a  focus  earlier  than  the  red  rays,  with 


FIG.  6. — Microscope  objectives   showing  the   component  parts  of   the   objective 
lens  system.     (After  Leitz.} 

the  remainder  of  the  spectrum  spread  out  between.  This  defect 
is  known  as  chromatic  aberration.  It  is  corrected  to  a  very  con- 
siderable extent  by  combining  biconvex  lenses  of  crown  glass 
with  plano-concave  lenses  of  flint  glass  (achromatic  objectives), 
to  a  still  nicer  degree  by  combinations  of  lenses  of  several  different 
kinds  of  glass  together  with  a  lens  of  fluorite  (apochromatic  ob- 
jectives); and  finally,  when  desired,  chromatic  aberration  may 
be  wholly  avoided  by  employing  mono-chromatic  light. 

A  third  defect  of  lenses  is  known  as  diffraction,  which  is  a 
phenomenon  giving  rise  to  a  whole  group  of  less  luminous  second- 
ary images  around  the  principal  image.  The  influence  of  dif- 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS 


21 


FIG.  7.  —  Sectional  view  of  a  compound  microscope  illustrating  the  course  of 

)  and  indic 
After  Leitz.} 


IG.  7.  —  econa    v 

two  beams  preceding  from  two  points  in  the  object  (P  and  Q)  and  indicating  the 
subjective  interpretation  of  the  image  formed  on  the  retina.     ( 


22  BACTERIOLOGY 

fraction  is  most  evident  when  the  surfaces  of  the  lens  are  rough- 
ened by  scratches  or  by  presence  of  dust,  but  even  the  most  per- 
fect lens  systems  are  not  wholly  free  from  diffraction  phenom- 
ena. Some  of  these  defects  will  require  brief  consideration  in 
our  discussion  of  the  compound  microscope. 

In  the  modern  compound  microscope  the  beam  of  light  pro- 
ceeding from  a  point  in  the  object  is  refracted  by  the  lens  system 


FIG.  8. — Image  formation  in  the  compound  microscope.     Compare  with  Fig.  9 

of  the  objective  (Fig.  6)  so  as  to  render  the  rays  slightly  conver- 
gent. Near  the  upper  end  of  the  tube  of  the  microscope  these 
rays  are  further  refracted  by  the  lower  lens  of  the  eye-piece  and  are 
converged  and  brought  to  a  focus  in  the  interior  of  the  eye-piece. 
A  screen  placed  at  this  level  would  show  a  real  image,  and  any 
pattern  (for  example  an  eye-piece  micrometer)  inserted  in  the 


FIG.  9. — Image  formation  in  the  compound  microscope  with  an  eye-piece  of 
higher  power.  Observe  that  the  increased  magnification  is  accomplished  by  narrow- 
ing the  beam  of  light  which  enters  the  eye  and  so  diminishing  the  size  of  the  closing 
angle.  Compare  with  Fig.  .8. 

eye-piece  at  this  level  is  readily  fused  with  the  microscopic  field. 
Continuing  in  a  straight  line  the  rays  diverge  from  this  focus  to 
reach  the  upper  lens  of  the  eye-piece.  In  traversing  this  lens 
they  are  again  refracted  and  made  parallel  so  that  they  will 
enter  the  eye  and  be  brought  to  a  focus  on  the  retina.  The  paths 
of  two  beams  of  light,  one  proceeding  from  the  center  of  the  mi- 
croscopic field  and  one  from  its  periphery,  are  illustrated  in  Fig. 


THE   MICROSCOPE   AND    MICROSCOPIC   METHODS  23 

8.  Fig.  9  shows  the  change  which  is  introduced  by  the  use  of 
an  eye-piece  of  higher  magnifying  power. 

It  will  be  noted  that  the  objective  and  lower  lens  of  the  eye- 
piece bring  the  beam  to  a  focus  forming  a  real  image,  and  that 
the  rays  diverging  again  from  this  image  are  again  brought  to  a 
focus  on  the  retina  by  the  upper  lens  of  the  eye-piece  and  the  op- 
tical structures  of  the  eye.  The  magnification  represented  in  the 
first  image  is  the  quotient  of  the  sine  of  the  angle  of  the  opening 
limb  of  the  beam  divided  by  the  sine  of  the  closing  angle.  The 
subsequent  magnification  between  this  and  the  eye  is  the  quotient 
of  the  sine  of  the  opening  angle  of  the  rays  proceeding  from  this 
image  divided  by  the  sine  of  the  closing  angle  of  the  rays  approach- 
ing the  retina.  The  closing  angle  at  the  formation  of  the  first 
image  and  the  opening  angle  of  the  beam  proceeding  from  it  are 
obviously  equal,  so  that  the  total  magnification  equals  the  sine 
of  the  first  opening  angle  divided  by  the  sine  of  the  last  closing 
angle  in  the  system.  It  will  be  noted  that  the  eye-piece  of  higher 
power  narrows  the  beam  and  decreases  the  closing  angle. 

In  the  above  discussion,  the  refractive  index  of  the  vitreous 
humor  has  been  disregarded.  This  is  not  the  same  as  that  of 
air  (in  reality  it  is  about  1.3)  and  the  peripheral  beam  is  there- 
fore bent  toward  the  axis  of  the  eye  instead  of  proceeding  in  its 
former  direction,  the  magnification  being  thereby  reduced  by 
precisely  the  fraction 


refractive  index  of  air 


—  or 


refractive  index  of  vitreous      1.3 

This  brings  us  to  a  definition  of  numerical  aperture.  The 
numerical  aperture  of  the  closing  limb  (n.a.)  is  the  sine  of  half 
the  angle  of  the  converging  beam  multiplied  by  the  refractive 
index  of  the  medium  (in  this  instance  the  vitreous  humor). 
This  is  commonly  designated  as  n.a.  The  numerical  aperture 
of  the  opening  limb  of  the  beam  (N.A.),  proceeding  from  a  point 
in  the  object  to  the  objective,  is  the  sine  of  half  the  angle  of  this 
beam  multiplied  by  the  refractive  index  of  the  medium  through 


BACTERIOLOGY 


which  it  passes.  This  is  commonly  designated  as  N.A.  Many 
desirable  properties  of  objectives,  other  than  magnification,  such 
as  brilliancy  of  illumination,  definition,  and  resolution  in  depth, 
also  depend  upon  the  numerical  aperture,  which  is  therefore 
perhaps  the  most  important  single  feature  of  objectives  of  high 
power. 


FIG.  io.— Central  il- 
lumination by  a  narrow 
beam.  Three  beams  of 
parallel  rays,  such  as 
might  come  from  a  large 
white  cloud,  are  repre- 
sented. Note  that  these 
rays  reach  the  object  as 
almost  vertical  rays, 
varying  from  the  vertical 
by  only  a  narrow  angle. 
Compare  with  Fig.  15. 


FIG.  1 1 . — Illumination 
by  a  hollow  cone  of  light 
converging  upon  the  object 
at  a  wide  angle,  by  use  of 
the  central  spot  stop. 
Compare  with  Fig.  14,  and 
with  Fig.  16. 


FIG.  1 2 . — Illumina- 
tion by  a  broad  beam 
converging  upon  the  ob- 
ject at  a  wide  angle. 
Only  a  few  beams  of  par- 
allel rays  from  a  distant 
point  source  of  light  are 
represented  in  the  figure. 
Compare  with  Fig.  17. 


Another  important  optical  part  of  the  bacteriological  micro- 
scope is  the  substage  illuminating  apparatus,  consisting  of  the 
mirror,  the  iris  diaphragm  and  the  condenser.  These  are  neces- 
sary to  illuminate  minute  objects  so  that  they  may  be  satis- 
factorily studied  at  high  magnifications.  By  the  use  of  the  iris 
diaphragm  and  of  the  central  spot  stop,  the  ordinary  condenser 
may  be  made  to  furnish  three  different  kinds  of  illumination,  (i) 


THE    MICROSCOPE    AND    MICROSCOPIC   METHODS  25 

central  illumination  by  a  narrow  beam,  (2)  illumination  by  a  hollow 
cone  of  light  converging  on  the  object  at  a  wide  angle,  an  ex- 
ample of  dark-field  illumination,  and  (3)  intense  illumination  by  a 
broad  beam  converging  at  a  wide  angle  upon  the  object.  These 
possibilities  are  illustrated  in  Figs.  10,  n  and  12.  Dark-field 
illumination  is  obtained  in  a  more  satisfactory  manner  by  em- 
ploying a  special  condenser  made  for  the  purpose,  illustrated  in 
Figs.  13  and  14.  The  way  in  which  these  different  methods  of 


FIG.  13. — Dark-field  condenser  showing  optical  FIG.  14. — Optical  parts  of 

parts  and  centering  mechanism.  (After  Leitz.}        the  dark-field  condenser  with 

object  slide  .and  microscope 
objective  with  funnel  stop  in 
position.  The  path  of  light 
rays  is  indicated  by  the  dotted 
lines.  (After  Leitz.) 

illumination  affect  the  visibility  of  a  colorless  refractive  object 
is  illustrated  in  Figs.  15,  1 6  and  17. 

Visibility  of  Microscopic  Objects.  — In  the  use  of  the  mi- 
croscope it  is  necessary  to  pay  some  attention  to  the  factors  upon 
which  visibility  depends.  An  object  may  be  distinguished  and 
perceived  by  the  eye  only  when  the  light  coming  from  the  object 
differs  from  that  coming  from  its  surroundings  either  in  quantity 
or  in  quality,  and  the  greater  the  extent  of  this  difference  the 


26  BACTERIOLOGY 

more  distinctly  visible  will  the  object  be.  Uncolored  trans- 
parent objects  are  visible  by  virtue  of  their  ability  to  refract  light 
and  so  to  present  darker  and  lighter  zones.  If  the  surrounding 
medium  possess  the  same  refractive  power  as  the  colorless  trans- 
parent object,  the  latter  is  invisible.1  Microscopic  objects  may 
conceivably  be  invisible  or  so  nearly  invisible  as  to  have  escaped 


FIG.  15. — Showing  the  manner  in  which  the  "dark  outline  picture"  is  produced. 

(After  A.  E.  Wright.) 

detection  for  this  very  reason.  If,  however,  the  object  be  sus- 
pended in  a  medium  of  lower  refractive  index,  then  it  may  be  de- 
nned by  light  and  shade,  and  it  is  most  clearly  defined  when  illu- 
minated in  one  of  two  ways,  either  by  a  rather  narrow  direct  beam 
of  light  passing  from  behind  it  directly  toward  the  eye,  in  which 
case  the  object  is  denned  by  dark  outlines  upon  a  white  field;  or  by 

1  This  may  be  illustrated  fairly  well  by  immersing  clean,  perfectly  clear  glass 
beads  in  oil  of  cedar  wood. 


THE   MICROSCOPE   AND   MICROSCOPIC   METHODS  27 


FIG.  16. — Showing  the  manner  in  which  the  "bright  outline  picture"  is  produced 

(After  A.  E.  Wright.} 


FIG.  17. — Showing  the  manner  in  which  the  outlines  are  obliterated  when  an  object 
is  illuminated  by  a  homogeneous  illuminating  field.     (After  A.  E.  Wright.} 


28  BACTERIOLOGY 

oblique  beams  directed  at  an  angle  from  the  sides,  when  the  object 
is  defined  by  bright  outlines  on  a  dark  background.  If,  how- 
ever, the  object  be  illuminated  from  all  sides  or  from  behind  and 
from  both  sides  by  light  of  similar  intensity,  its  outlines  become 
less  distinct  and  may  even  be  completely  obliterated  so  that  the 
object  becomes  invisible.  These  facts  may  be  crudely  illustrated 
by  holding  a  test-tube  full  of  water,  (i)  between  the  eye  and  a 
window,  (2)  between  the  eye  and  a  dark  wall  between  two  win- 
dows, and  (3)  against  the  center  of  the  window  pane.  Their 
importance  in  microscopy  may  be  readily  illustrated  by  examining 
a  simple  preparation  of  living  bacteria,  (i)  with  the  iris  diaphragm 
nearly  closed,  (2)  with  the  dark-field  condenser,  and  (3)  with  the 
ordinary  condenser  with  the  iris  wide  open.  It  will  be  evident 
that  the  third  arrangement  is  fatal  to  the  definition  of  colorless 
transparent  microscopic  objects.  It  will  also  be  observed  that 
the  dark  field  offers  an  advantage  in  the  ease  with  which  the  ob- 
jects can  be  seen,  the  small  luminous  outline  on  the  dark  back- 
ground being  more  distinct  then  the  dark  outline  on  the  luminous 
background.  The  former  might  be  compared  in  this  respect 
to  a  star  at  night,  and  the  latter  to  a  sun  spot  in  the  day- 
time, which  though  many  times  larger  may  not  be  readily 
perceived. 

The  method  of  making  objects  visible  by  a  difference  in 
quality  of  light  (color)  usually  involves  the  necessity  of  staining. 
Colored  preparations  have  certain  very  important  advantages 
for  microscopic  study.  If  an  object  can  be  differentially  colored, 
that  is,  stained  a  different  color  or  a  different  shade  of  the  same 
color  from  the  material  by  which  it  is  surrounded,  it  becomes 
clearly  visible  even  in  the  absence  of  different  refractive  power. 
Refraction  may  be  largely  eliminated  by  replacing  the  fluids  of 
the  preparation  by  other  fluids  of  high  refractive  index,  such  as 
cedar  oil  or  balsam,  and  this  elimination  of  refraction  eliminates 
the  opacity  of  the  preparation,  "  clears"  it,  and  makes  possible  the 
distinct  definition  of  minute  objects  situated  in  the  deeper  optical 
planes  of  the  preparation.  A  proper  appreciation  of  this  mi- 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        2Q 

croscopical  principle  will  at  once  suggest  the  importance  of  differ- 
ential staining  methods  in  microscopy. 

The  Bacteriological  Microscope. — The  bacteriological  micro^ 
scope  consists  of  a  tubular  body  which  carries  the  optical  parts,  and 


FIG.  1 8. — Microscope. 

which  can  be  raised  or  lowered  for  focusing.  The  objectives  should 
be  three  in  number,  and  should  be  attached  to  the  body  by  means 
of  a  triple  nose-piece,  which  permits  any  objective  to  be  turned  into 
the  optical  axis  at  will.  The  eye-piece  slips  into  the  upper  and 
opposite  end  of  the  body  or  tube.  The  arrangements  for  focus- 


30  BACTERIOLOGY 

ing  consist  of  a  rack  and  pinion  which  accomplish  the  coarse  ad- 
justment, and  a  more  delicate  fine  adjustment.  The  stage,  upon 
which  the  objects  to  be  examined  are  placed,  has  an  opening  in 
the  middle.  In  this  opening  an  iris  diaphragm  and  Abbe  con- 
denser are  inserted.  The  iris  diaphragm  enables  one  to  alter 
the  size  of  the  opening  as  desired.  Beneath  the  stage  is  a  mov- 
able mirror,  of  which  one  side  is  plane  and  the  other  concave. 
All  of  these  parts  are  supported  on  a  short,  heavy  pillar  which 
is  fixed  in  the  horseshoe-shaped  base. 

The  essential  parts  of  the  microscope  are,  of  course,  the  eye- 
piece (German,  Ocular),  and  the 
objective.  Objectives  are  given 
various  names  by  different  makers, 
for  instance,  A,  B,  C,  etc.,  or  i,  2, 
3,  etc.;  or  they  are  named  accord- 
ing to  their  focal  distances,  as  f 

FIG.  19.  inch,  J  inch,  f  inch,  etc.      In  bac- 

Abbe  Condenser.     On  the  right  side  the,      .   ,      .      , 

figure  gives  a  sectional  view.       tenological   work   a   rather     low 

power"  |  or  f  inch  objective,  an 

ordinary  "high  power"  J  to  f  inch  dry  objective,  and  a  high  power 
-jV  inch  oil-immersion  objective  are  needed.  The  magnification 
with  the  f  or  f  inch  objective  is  about  75  to  100  diameters;  with 
the  J  to  |  inch  400  to  700  diameters;  with  the  yV  immersion 
750  to  1,000  diameters.  The  magnification  varies  according  to 
the  eye-piece  used,  as  well  as  with  the  objective.  A  i  inch  and 
if  inch  eye-piece  (Leitz  No.  2  and  No.  4)  serve  well  for  most 
purposes.  The  eye-pieces  are  Usually  named  arbitrarily,  like  the 
objectives.  The  oil-immersion  objective  is  used  in  the  exami- 
nation of  bacteria  where  a  very  high  power  is  desired.  A  layer 
of  thickened  oil  of  cedar-wood  is  placed  between  the  lower  sur- 
face of  the  objective  and  the  upper  surface  of  the  glass  covering 
the  object  under  examination.  The  oil  must  be  wiped  away 
from  the  surface  of  the  objective  when  the  examination  is  finished. 
For  this  purpose  the  soft  paper  sold  by  dealers  in  microscopical 
apparatus  serves  admirably.  Care  must  be  taken  not  to  scratch 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        31 

the  lower  surface  of  this  objective.  Oil  of  cedar- wood  furnishes 
a  medium  having  nearly  the  same  refractive  index  as  the  glass  of 
the  lens  and  the  glass  on  which  the  object  is  mounted,  and  it  ob^ 
viates  the  dispersion  of  light  which  takes  place  when  a  layer  of 
air  is  interposed  between  the  objective  and  the  object,  as  happens 
with  the  ordinary  dry  lens. 

The  microscope  should  be  placed  in  front  of  the  observer  on 
a  firm  table.  The  observer  should  be  able  to  bring  the  eye  easily 
over  the  eye-piece  when  the  tube  of  the  microscope  is  in  vertical 
position.  Daylight  should  be  employed  if  possible.  When  arti- 
ficial illumination  is  necessary,  an  ordinary  lamp,  a  Welsbach 
burner  or  an  incandescent  electric  light  may  be  used.  It  is  best 
to  modify  the  artificial  light  by  inserting  a  sheet  of  blue  glass  be- 
tween the  light  and  the  mirror. 

In  order  to  focus  upon  any  object,  having  first  secured  a  satis- 
factory illumination  with  the  mirror,  it  is  best,  beginning  with 
the  low  power  and  using  the  coarse  adjustment  for  focusing,  to 
bring  the  objective  quite  close  to  the  object,  and  then,  with  the 
eye  in  position,  to  raise  the  tube  until  the  object  comes  into 
focus.  The  exact  focusing  is  done  with  the  fine  adjustment. 
The  observer  should  keep  both  eyes  open  when  using  the  micro- 
scope, and  should  be  able  to  use  either  eye  at  will. 

All  measurements  of  microscopic  objects  are  expressed  in 
terms  of  a  micromillimeter.  This  is  one-thousandth  of  a  milli- 
meter (o.ooi  mm.),  which  is  about  -^-^^-Q-Q  of  an  inch.  This  unit 
is  designated  as  a  micron,  and  is  denoted  by  the  Greek  letter  /*. 
For  example,  5  /*  =  0.005  mrn- =  5~oV<r  inch. 

The  Platinum  Wire. — The  substance  under  examination 
is  usually  placed  upon  thin  slips  of  glass  called  cover-glasses.  The 
material  is  spread  over  the  cover-glass  by  means  of  a  platinum 
wire  which  has  been  fixed  in  a  glass  rod  about  six  inches  long. 
Such  a  platinum  wire  is  used  constantly  in  doing  bacteriolog- 
ical work.  The  platinum  wire  must  be  stiff  enough  not  to 
bend  too  easily,  and  yet  it  should  not  be  so  large  that  it  will 
not  cool  rapidly  after  heating.  A  good  size  for  most  pur- 


BACTERIOLOGY 


O 


poses  is  No.  28,  English   standard  gauge,   diameter   .014  inch. 
The  wire  may  be  straight  throughout  its  length,  or  the  tip  may 
be  bent  to  form  a  loop  (German,  Oese).     It  is  well  to  follow,  from 
the  beginning,  certain  rules  which  make  the  use  of  the  platinum 
wire  safe  and  accurate.     Every  time  it  is  taken  into  the  hand 
and  before  using  it  for  any  manipulation,  heat  it 
in  the  flame  of  a  Bunsen  burner  or  an  alcohol  lamp 
to  a  red  heat;  and  always,  after  using  and  before 
putting  it  down,  heat  it  again  to  a  red  heat.     After 
the  needle  has  become  wet  by  dipping  it  in  a  fluid 
and  is  to  be  sterilized  in  the  flame,  it  is  necessary 
to  avoid  " sputtering"  of  the  fluid  by  bringing  the 
wet  needle  gradually  to  the  flame,  so  as  to  dry  the 
material  adhering  to  it  before  burning  it.     This 
procedure  must  be  done  with  great  care  when  the 
wire  has  been  dipped  in  milk  or  other  substances 
containing  oil.     When  the  needle  "  sputters,"  as 
it  is  called,  from  too  rapid  heating,  particles  that 
have  not  yet  been  sterilized  may  be  thrown  some 
distance.     On  no  account  should  the  needle  touch 
any  object  other  than  that  which  it  is  intended  it 
should  touch.     With  such  a  platinum  wire,  which 
has  been  properly  sterilized,  one  can  easily  remove 
portions  from  a   culture   of  bacteria,  or  from  a 
fluid  in  which  bacteria  are  supposed  to  be  present. 
The  glass  rod  in  which  the  platinum  wire  is  fixed 
should  be  held  between  the  thumb  and  forefinger  of  the  right 
hand  like  a  pen. 

-  Glass  Pipettes. — Sterile  glass  tubes  drawn  out  to  form  slender 
capillaries,  Pasteur  pipettes,  are  very  convenient  instruments 
for  handling  bacteriological  materials,  and,  for  many  kinds 
of  work,  really  indispensible.  They  serve  nearly  all  the  pur- 
poses of  the  platinum  wire  and  are  capable  further  of  use  to 
transfer  large  quantities  of  fluid  without  contamination.  They 
are  also  especially  useful  in  collecting  material  from  patients 


FIG.  20. — Need- 
les used  for  inocu- 
lating media. 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        33 

and  at  autopsy.     Each  pipette  is  sterilized  and  discarded  after 

use. 

These  pipettes  are  made  by  cutting  glass  tubing  of  a  suitable 
size,  diameter  3  mm.  to  9  mm.,  into  pieces  from  20  to  40  cm.  in 
length.  The  cut  ends  are  smoothed  in  the  flame.  In  the  tubes 
of  larger  caliber  it  is  well  to  make  a  constriction  about  5  cm.  from 
each  end.  Each  end  is  plugged  with  cotton.  The  tubes  are  then 
sterilized  by  dry  heat.  By  heating  the  middle  of  the  tube  in 
a  blast  lamp  or  over  a  large  Bunsen  flame,  the  glass  may  be 
softened  and  then  drawn  out  into  a  capillary  of  any  desired 


FIG.  21. — Drawn-out  tube  pipettes  of  Pasteur,  a,  Plugged,  sterile  tube  as 
kept  in  stock;  b,  the  same  heated  at  x  in  blast-lamp  and  drawn  out;  then  sealed  at 
x;  c  and  d,  completed  pipettes;  e,  the  same  with  bulb.  (After  Novy.) 

length  and  caliber.  This  is  melted  in  the  middle  and  severed 
by  the  flame,  giving  two  pipettes.  When  a  large  capacity  is 
desired  a  bulb  may  be  blown  in  the  tube  between  the  capillary 
and  the  cotton  plug.  This  requires  a  little  practice.  The  tip 
of  the  pipette  is  finally  broken  off  with  aid  of  a  file,  sterilized  by 
the  flame  and  the  pipette  is  ready  for  use.  The  various  steps  in 
the  preparation  of  pipettes  are  illustrated  in  the  figures  (Fig.  21). 

The  Hanging-drop. — Living  bacteria  may  be  studied  with 
the  microscope  while  suspended  in  some  fluid  substance.  The 
platinum  loop  having  been  heated  to  a  red  heat  in  the  flame  and 
having  been  allowed  to  cool,  a  small  portion  of  the  culture  or 

3 


34  BACTERIOLOGY 

other  material  may  be  removed  with  it  and  deposited  in  the  center 
of  an  ordinary  cover-glass.  The  needle  should  again  be  sterilized 
in  the  flame.  When  cultures  on  solid  media  are  to  be  examined, 
a  small  particle  may  be  mixed  with  a  drop  of  sterilized  water  or 
bouillon.  The  cover-glass  should  have  been  carefully  cleaned  and 
sterilized  over  the  flame.  The  cover-glass  with  the  small  drop 
of  fluid  material  held  in  sterilized  forceps  is  now  to  be  inverted 
over  a  sterilized  glass  slide,  which  has  a  concavity  ground  in  the 
middle  of  it.  Around  the  concavity,  the  slide  should  be  smeared 
with  vaseline.  In  this  manner  a  small  air-tight  chamber  is  made. 
This  slide  and  cover-glass  is  next  put  upon  the  stage  of  the  micro- 
scope. A  good  dry  lens,  if  of  sufficiently  high  power,  is  more 
convenient  for  examining  the  hanging-drop  than  an  oil-immer- 
sion. If  the  latter  be  used,  having  placed  a  drop  of  cedar-oil  on 
the  center  of  the  cover-glass,  and  a  good  light  having  been 


FIG.  22. 

secured,  the  oil-immersion  objective  should  be  brought  down 
upon  this  drop  of  oil.  The  beginner  often  experiences  difficulty 
in  focusing  upon  a  hanging-drop.  It  is  necessary  to  shut  off 
most  of  the  light  by  means  of  the  iris  diaphragm,  for  as  has 
already  been  pointed  out  (page  28),  colorless  objects  may  be  clearly 
seen  only  when  illuminated  either  by  a  narrow  central  beam  or 
by  oblique  illumination  (dark-field).  Often  it  is  well  to  secure 
the  focus  roughly  upon  the  extreme  outer  edge  of  the  chamber, 
or  to  find  the  edge  of  the  drop  of  fluid  with  the  low  power  and 
then  focus  upon  this  edge  with  the  oil-immersion  objective. 
Above  all  things  guard  against  breaking  the  cover-glass  by  forcing 
the  objective  down  upon  it.  The  motility  of  certain  bacteria  is  one 
of  the  most  striking  phenomena  to  be  observed  in  the  hanging- 
drop.  It  is  not  to  be  confused  with  the  so-called  "Brownian 
movement' '  which  is  exhibited  by  fine  particles  suspended  in  a 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        35 

watery  fluid.  It  is  well  for  the  beginner  to  observe  the  character 
of  the  Brownian  movement  by  rubbing  up  some  carmine  in^a- 
little  water,  and  with  the  microscope  to  study  the  trembling 
motion  exhibited  by  these  particles  of  carmine.  It  will  be  noticed 
that,  although  the  particles  oscillate,  no  progress  in  any  direction 
is  accomplished  unless  there  are  currents  in  the  fluid.  Such  cur- 
rents might  give  rise  to  the  impression  that  certain  bacteria 
possessed  motility  when  they  were,  in  fact,  powerless  to  move 
of  themselves.  In  the  hanging-drop  the  multiplication  of  bacteria 
can  be  studied,  the  formation  of  spores  and  the  development  of 
spores  into  fully  formed  bacteria.  The  hanging-drop  is  also 
used  extensively  for  the  demonstration  of  the  agglutination 
reaction  with  the  bacillus  of  typhoid  fever.  Sometimes  bacteria 
must  be  watched  in  the  hanging-drop  for  hours,  or  even  days, 
and  it  may  be  necessary  to  keep  it  at  the  temperature  of  the 
human  body  for  this  length  of  time.  Various  complicated  kinds 
of  apparatus  have  been  devised  for  this  purpose,  but  they  are 
needful  only  for  special  kinds  of  work.  When  the  hanging- 
drop  preparation  is  no  longer  required,  the  slide  and  cover-glass 
should  be  dropped  into  a  5  per  cent  carbolic  acid  solution  and 
afterward  sterilized  by  steam. 

The  Hanging-block. — Hanging-block  preparations,  which 
were  introduced  by  Hill,1  make  use  of  a  cube  of  nutrient  agar 
instead  of  a  drop  of  fluid.  Bacteria  are  distributed  on  the  sur- 
face of  the  agar,  which  is  then  applied  to  a  cover-glass,  and 
mounted  like  a  hanging-drop.  The  bacteria  are  thus  kept  in  a 
layer  close  to  the  glass,  where  growth  may  be  studied. 

The  Microscopic  Preparation  for  Study  by  Dark-field  Illumi- 
nation.— The  central  portion  of  a  clean  glass  slide  is  encircled 
with  a  ring  of  vaseline,  and  a  drop  of  the  fluid  to  be  examined 
is  deposited  on  the  clean  surface  in  the  center  of  the  ring  by  means 
of  a  capillary  tube.  It  is  then  covered  with  a  clean  large  cover- 
glass  so  that  the  fluid  spreads  out  in  a  moderately  thin  layer 
beneath  the  cover-glass  and  is  confined  on  all  sides  by  the 

1  Journal  of  Medical  Research,  Vol.  VII.,  March,  1902. 


36  BACTEEIOLOGY 

vaseline,  thus  preventing  evaporation  and  resulting  currents  in 
the  preparation. 

Best  results  with  the  dark-field  microscope  are  obtained 
only  in  a  dark  or  dimly  lighted  room.  An  electric  arc  or  a  power- 
ful gas-light  may  be  employed  as  the  source  of  light,  and  it  is  well 
to  put  a  flask  of  water  between  the  light  and  the  microscope  to 
eliminate  the  heat-rays.  The  substage  condenser  of  the  micro- 
scope is  replaced  with  the  special  dark-field  condenser  and  this 
is  carefully  centered.  A  large  drop  of  immersion  oil  is  placed  on 
the  upper  surface  of  the  condenser.  The  slide  is  carefully  placed 
upon  the  stage  so  that  the  oil  fills  in  completely  the  space  between 
the  condenser  and  slide  and  remains  free  from  air  bubbles. 
The  preparation  is  then  ready  for  examination.  Objectives  of 
numerical  aperture  wider  than  i.o  cannot  be  successfully  used 
with  the  ordinary  dark-ground  condensers  and  therefore  it  is 
necessary  to  stop  down  the  aperture  of  the  oil-immersion  objec- 
tive before  using  it.  A  special  funnel  stop  is  furnished  for  this 
purpose.  When  this  has  been  "attached  the  preparation  may  be 
studied  with  the  oil-immersion  objective  in  the  usual  way.  Skill 
in  this  method  of  studying  unstained  microbes  is  quickly  acquired, 
offering,  as  a  rule,  less  difficulty  than  the  method  of  central 
illumination  which  is  employed  for  the  hanging- drop  and  hanging- 
block. 

Smear  Preparations  for  Staining. — The  examination  of 
bacteria  with  the  microscope  is  carried  out  to  a  very  large  extent 
by  means  of  smears  made  upon  thin  slips  of  glass.  Such  slips 
of  glass  are  generally  called  cover-glasses.  It  is  best  to  obtain  the 
kind  sold  by  dealers  as  No.  i,  f  inch  squares. 

The  cover-glass  may  be  cleaned  best  by  immersion  in  a  mix- 
ture of  sulphuric  acid  and  bichromate  of  potassium  solution,  and 
afterward  washed  thoroughly  in  distilled  water,  and  finally  in 
alcohol.  A  stock  of  clean  cover-glasses  may  be  kept  in  a  bottle 
of  alcohol,  or  perhaps  preferably  in  alcohol  containing  3  per  cent 
of  hydrochloric  acid. 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS  37 

CLEANING  FLUID. 

Potassium  bichromate 40  grams. 

Water 150  c.c. 

Dissolve  the  bichromate  of  potassium  in  the 
water,  with  heat;  allow  it  to  cool;  then  add 
slowly  and  with  care  sulphuric  acid,  com- 
mercial   230  c.c. 

When  they  are  needed  for  use  they  should  be  wiped  clean 
with  a  piece  of  linen  cloth.  As  a  rule,  cover-glasses  cleaned  in 
this  way  still  retain  a  small  amount  of  oily  matter  on  their  surfaces, 
sufficient  to  prevent  the  proper  spreading  of  a  drop  of  water. 
This  difficulty  may  be  overcome  by  passing  each  glass  several 
times  through  the  flame.  It  is  better,  when  time  permits,  to  fill 
an  Esmarch  dish  with  clean  cover-glasses  and  then  heat  them  in 
the  oven  at  200°  C.  for  half  an  hour.  Cover-glasses  treated  in  this 
way  will  allow  the  droplet  of  bacterial  suspension  or  other  material 
to  spread  perfectly.  They  must  be  carefully  preserved  in  a 
covered  dish  from  which  they  are  to  be  removed  only  by  clean 
(flamed)  forceps.  Carelessness  in  this  matter  may  necessitate 
recleaning  of  the  whole  lot  of  cover- glasses. 

An  ordinary  pair  of  fine  forceps  may  be  used  to  pick  up  the 
cover-glass  and  insert  it  between  the  blades  of  such  special  forceps 
as  those  of  Cornet  or  of  Stewart.  Perhaps  the  most  convenient 
style  of  forceps  is  that  devised  by  Novy,  provided  with  a  clasp. 
Bacteria  may  be  placed  upon  the  cover-glass  by  allowing  the 
glass  to  fall  upon  one  of  the  colonies  of  bacteria,  on  a  gelatin  or 
agar  plate  (see  page  no),  which  will  adhere  to  it  in  part,  produc- 
ing an  "  impression  preparation"  (German,  Klatschpreparat). 
Such  a  preparation,  after  drying  in  the  air,  is  to  be  fixed  by  pass- 
ing it  through  the  flame  three  times.  (See  below.)  The  forceps 
with  which  it  is  handled  should  be  sterilized  in  the  flame. 

Generally  bacteria  contained  in  fluids,  like  sputum,  or  taken 
from  the  surface  of  a  culture,  are  smeared  over  the  cover-glass 
by  means  of  the  platinum  wire  or  loop,  which  must  be  heated  to 
a  red  heat  before  and  after  the  operation.  Such  preparations 


BACTERIOLOGY 


are  called  smear,  cover-glass,  cover-slip,  or  film  preparations. 
When  the  material  to  be  spread  is  thick  or  very  viscid,  a  small 
drop  of  distilled  water  must  first  be  placed  in  the  center  of  the 
cover-glass  so  as  to  dilute  it.  Beginners  generally  take  too  much 


FIG.  23. — Cornet  forceps  for  cover-glasses. 

material  on  the  wire.  As  thin  a  smear  as  possible  is  made.  It 
is  allowed  to  dry  in  the  air;  this  should  occupy  a  few  seconds. 
The  drying  may  be  hastened  4by  holding  the  forceps  with  the 
cover-glass  a  long  distance  above  the  flame,  at  a  point  where 
the  heat  would  cause  no  discomfort  to  the  hand.  Having  dried 


FIG.  24. — Stewart  forceps  for  cover-glass. 

the  preparation,  it  is  to  be  passed  through  the  flames  of  a  Bunsen 
burner  or  alcohol  lamp  three  times,  taking  about  one  second  for 
each  transit.  The  heat  of  the  flame  serves  to  dry  the  bacteria 
upon  the  cover-glass  and  fix  them  permanently  in  position;  it  is 
not  sufficient,  however,  when  applied  in  this  manner,  to  kill  all 


FIG.  25. — Novy's  cover-glass  forceps  withe  clasp.     (After  Novy.) 

kinds  of  bacteria,  especially  those  containing  spores.  After  it 
has  been  passed  through  the  flame  three  times  the  preparation 
may  be  stained  with  one  of  the  aniline  dyes,  and  after  washing 
in  water  and  drying,  may  be  mounted,  face  down,  in  Canada 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS 


39 


balsam  upon  a  glass  slide.  It  makes  a  suitable  object  to  be 
examined  with  the  oil-immersion  objective.  The  slide  is  a  thin 
slip  of  glass,  3  inches  by  i  inch,  with  ground  edges. 

The  smear  preparation  may  equally  well  be  made  directly 
upon  the  glass  slide  provided  this  be  cleaned  and  heated  to  insure 
a  clean  surface  free  from  oily  matter.  The  fixation  in  the  flame 
must  then  occupy  a  longer  time  than  with  the  small  and  thin 
cover-glass.  Such  preparations  have  the  advantage  that  several 
may  be  made  upon  one  slide,  and  that  after  staining  them  they 
may  be  examined  in  cedar-oil,  with  the  oil-immersion  lens, 
without  the  use  of  the  cover-glass  and  Canada  balsam.  They 
are  also  less  readily  broken  in  handling.  The  forceps  of  Kirk- 
bride  will  be  found  convenient  when  staining  on  the  slide.  The 


FIG.  26. — Kirkbride  forceps  for  holding  slides. 

aluminium  dish  devised  by  Krauss,1  or  some  similar  dish,  will 
be  found  useful  when  the  stain  has  to  be  heated.  Experiments 
have  shown  that  the  ordinary  method  of  fixation  in  the  flame, 
when  applied  to  bacteria  spread  upon  slides,  has  little  effect  on 
the  vitality  of  many  species.  The  beginner  is,  therefore,  advised 
to  make  his  preparations  on  cover-glasses. 

When  very  resistant  or  dangerous  pathogenic  bacteria  are 
being  handled,  after  fixation  by  heat  upon  the  slide  or  cover- 
glass,  the  preparation  may,  if  desired,  be  immersed  in  i-iooo 
solution  of  bichloride  of  mercury  long  enough  to  kill  the  bacteria, 
without  injuring  the  preparation  or  its  staining  properties. 

Staining  Solutions. — The  staining  of  bacteria  is  done  for  the 
most  part  with  the  aniline  dyes.  The  object  of  staining  bacteria 
is  to  give  them  artificially  some  color  which  makes  them  distinct 

1  Krauss,  Jour.  A.M.  A.,  Apr.  6,  1912,  Vol.  LVIII,  p.  1013. 


4O  BACTERIOLOGY 

and  easily  visible  without  imparting  this  color  to  the  substance 
or  medium  in  which  they  are  imbedded.  The  substances  known 
as  aniline  dyes  are  derivatives  of  coal-tar,  but  not  always  of  ani- 
line. These  dyes  are  of  great  importance  in  bacteriological 
work.  Their  number  is  very  large,  but  only  a  few  are  in  common 
use.  It  is  important  to  have  the  purest,  and  those  obtainable 
from  Griibler  are  reliable. 

It  is  simplest  to  classify  the  aniline  dyes  as  acid  or  basic. 
Eosin,  picric  acid  and  acid  fuchsin  are  acid  dyes;  they  tend  to  stain 
tissues  diffusely.  Fuchsin,  gentian-violet  and  methylene  blue  are 
basic  dyes;  they  have  an  affinity  for  the  nuclei  of  tissues  and  for 
bacteria;  they  therefore  are  the  dyes  used  chiefly  in  bacteriolog- 
ical work.  The  other  varieties  may  be  employed  as  contrast- 
stains;  another  contrast-stain  frequently  used  is  Bismarck  brown. 
It  is  best  to  keep  on  hand  saturated  solutions  of  the  aniline  dyes 
in  alcohol,  which  are  permanent,  but  cannot  be  employed  directly 
for  staining.  In  order  to  prepare  the  simple  staining  solutions, 
the  alcoholic  solution  is  diluted  about  ten  times,  or  so  as  to  make 
a  liquid  which  is  just  transparent  in  a  layer  about  12  mm.  in 
thickness,  after  filtering.  These  watery  solutions  deteriorate 
after  a  few  weeks. 

Fuchsin  and  gentian-violet  stain  rapidly  and  intensely. 
Methylene  blue  works  more  slowly  and  feebly;  it  is  to  be  pre- 
ferred where  the  bacteria  occur  in  thick  or  viscid  substances, 
like  pus,  mucus,  and  milk. 

Aniline-water  Staining  Solutions. — The  intensity  with  which 
aniline  dyes  operate  may  be  increased  by  adding  aniline  oil  to 
the  solution: 

Aniline  oil 5  c.c. 

Water 100  c.c. 

Mix,  shake  vigorously,  filter  through  wet  filter  paper.     The  fluid 
after  filtration  should  be  perfectly  clear.     Add— 

Alcohol 10  c.c. 

Alcoholic  solution  of  fuchsin  (or  gentian  violet,  or 

methylene  blue) i  c.c. 


THE    MICROSCOPE    AND    MICROSCOPIC   METHODS  41 

Aniline-water  staining  solutions  do  not  keep  well,  and  need  to 
be  freshly  prepared  about  every  two  weeks.  The  applications 
of  the  aniline-water  stains  will  be  given  under  separate  headings7 
In  general,  however,  they  are  employed  where  a  stain  of  unusual 
power  is  required. 

Carbol-fuchsin. — The  intensity  of  staining  may  also  be  in- 
creased by  the  presence  of  carbolic  acid.  The  most  common 
example  of  this  is  carbol-fuchsin. 

Saturated  alcoholic  solution  of  fuchsin 10  c.c. 

5  per  cent  aqueous  solution  carbolic  acid 100  c.c. 

This  solution  keeps  for  some  months.  It  is  employed  especi- 
ally where  very  intense  action  is  required,  as  in  staining  spores, 
flagella,  and  acid-proof  bacteria. 

Loffler's  Methylene  Blue. — A  very  useful  solution,  which 
keeps  well,  isLoffler's  alkaline  methylene  blue: 

Saturated  alcoholic  solution  of  methylene  blue.  .     30  c.c. 
1-10,000  aqueous  solution  of  potassium  hydroxide  100  c.c. 

This  solution  stains  more  intensely  than  simple  methylene  blue, 
and  also  gives  rise  to  useful  differential  staining  in  smears  and 
even  in  sections  of  tissue. 

Nocht-Romanowsky  Stain. — This  requires  two  solutions,  one 
of  ripened  alkaline  methylene  blue,  the  other  of  eosin. 

Solution  i. 

Methylene  blue i .  o  gram. 

Sodium  carbonate 0.5  gram. 

Distilled  water 100.0  grams. 

Heat  at  60°  C.  for  two  days  until  solution  shows  a  slight  purplish 
color. 

Solution  2. 

Eosin,  yellowish,  water  soluble i  .o  gram. 

Distilled  water 100.0  c.c. 

In  staining,  a  few  drops  of  each  of  these  solutions  are  mixed  with 
about  10  c.c.  of  distilled  water  in  an  Esmarch  dish,  and  the  smear, 


42  BACTERIOLOGY 

which  has  previously  been  fixed  in  absolute  methyl  alcohol,  is 
floated  on  this  mixture  for  about  ten  minutes.  Considerable 
practice  is  necessary  before  the  best  results  are  obtainable. 
The  method  is  especially  useful  in  staining  blood  films,  and 
protozoa  in  blood,  in  feces  or  in  culture. 

Leishman's  Stain. — Leishman  has  utilized  the  principle  of 
Jenner's  stain1  and  has  added  to  it  the  important  additional 
constituents  found  in  polychrome  methylene  blue  by  substituting 
this  for  the  ordinary  methylene  blue  used  by  Jenner. 

Solution  A. — To  a  i  per  cent  solution  of  medicinally  pure 
methylene  blue  in  distilled  water  add  0.5  per  cent  sodium  car- 
bonate and  heat  at  65°  C.  for  12  hours,  then  allow  it  to  stand  10 
days  at  room  temperature. 

Solution  B. — Eosin  extra  B.  A.  (Griibler)  o.i  per  cent  solution 
in  distilled  water. 

Mix  Solutions  A  and  B  in  equal  amounts  and  allow  to  stand 
six  to  twelve  hours,  stirring  at  intervals.  Filter  and  wash  the 
precipitate  thoroughly.  Collect,  dry  and  powder  it.  0.15  gram 
is  dissolved  in  100  c.c.  of  pure  methyl  alcohol  to  form  the  staining 
solution.  It  keeps  perfectly  for  at  least  five  months.  To  stain, 
cover  the  dried  but  unfixed  film  of  blood  with  the  staining  solu- 
tion. After  30  to  60  seconds  add  about  an  equal  amount  of 
distilled  water.  Allow  this  mixture  to  act  for  five  minutes. 
Wash  in  distilled  water  for  about  one  minute,  examining  the 
specimen  mounted  in  water  under  the  microscope.  Blot,  dry 
thoroughly,  mount  in  balsam,  or  preserve  the  specimen  as  an 
unmounted  film. 

Numerous  imitations  or  modifications  of  Leishman's  stain 
have  been  described. 

Giemsa's  Stain. — This  stain  contains  certain  of  the  essential 
constituents  of  polychrome  methylene  blue  and  eosin,  the  whole 
being  dissolved  in  a  mixture  of  glycerin  and  methyl  alcohol. 
Giemsa's  Azur  I  is  the  substance  methylene  azure  and  his  Azur 

1  Jenner  (Lancet,  1899,  I,  p.  370)  first  employed  the  solution  of  eosin  and  methy- 
lene blue  in  methyl  alcohol  as  a  stain  for  blood  films. 


THE   MICROSCOPE   AND    MICROSCOPIC   METHODS  43 

II  is  this  substance  mixed  with  an  equal  amount  of  methylene 
blue.  His  Azur  II-eosin  is  the  compound  precipitated  when 
aqueous  solutions  of  Azur  II  and  eosin  are  mixed.  The  Giemsa 
solution  is  made  according  to  the  following  formula : 

Azur  II-eosin 3.0  grams. 

Azur  II 0.8  gram. 

Glycerin 250 .  o  grams. 

Methyl  alcohol 250.0  grams. 

Dissolve  the  powdered  dyes  in  the  glycerin  at  60°  C.;  then  add 
the  methyl  alcohol  previously  heated  to  the  same  temperature. 
After  mixing,  let  it  stand  24  hours  at  room  temperature,  and 
filter.  To  stain,  mix  one  drop  of  this  solution  with  i  c.c.  of  water 
and  immerse  the  film,  previously  fixed,  for  15  minutes  to  24  hours. 
Direct  preparation  of  Romanowsky  Stains. — In  a  study  of  the 
essential  constituents  of  the  Romanowsky  stain,  MacNeal1 
found  both  methylene  azure  and  methylene  violet  to  be  present 
and  participating  in  the  nuclear  staining.  The  preparation  of 
solutions  directly  from  the  pure  dyes,  methylene  azure,  methy- 
lene violet,  methylene  blue  and  eosin,  has  been  recommended 
as  the  best  manner  of  preparing  these  staining  solutions,  as  the 
proportion  of  the  various  constituents  may  be  varied  at  will  to 
obtain  various  kinds  of  differentiation.  As  a  routine  blood 
stain  for  study  of  leukocytes  and  staining  of  hematozoa,  the  fol- 
lowing is  recommended : 

Solution  A . 

Methylene  azure 0.3 

Methylene  violet  (Bernthsen's,  insoluble  in  water) .  o.  i 

Methylene  blue 2.4 

Methyl  alcohol,  pure 500.  o 

Solution  B. 

Eosin,  yellowish,  water  soluble 2.5 

Methyl  alcohol,  pure 500 .  o 

These  solutions  keep  for  at  least  a  year.  They  are  mixed  in  equal 
parts  and  diluted  by  the  addition  of  25  c.c.  of  methyl  alcohol  to 

1  Journ.  Infectious  Diseases,  Vol.  Ill,  1906,  pp.  412-433. 


44  BACTERIOLOGY 

each  ioo  c.c.  of  the  mixture.  This  final  mixture  is  employed 
in  the  same  manner  as  Leishman's  stain.  It  keeps  for  a  few 
months. 

Method  of  Staining  Cover-glass  Preparations. — (a)  A  smear 
preparation  of  bacteria  having  been  made  and  fixed  in  the  manner 
above  described,  and  a  watery  solution  of  either  fuchsin,  gentian 
violet  or  methylene  blue  having  been  prepared,  the  cover-glass 
is  to  be  dropped  into  a  dish  containing  the  dye,  or  the  dye  may 
be  dropped  upon  the  cover  glass  held  in  the  forceps. 

(b)  Allow  the  stain  to  act  for  about  thirty  seconds. 

(c)  Wash  in  water. 

(d)  Examine  with  the  microscope  in  water  directly  or  after 
drying  and  mounting  in  Canada  balsam. 

The  rapidity  and  intensity  of  staining  may  be  increased  by 
warming  the  solution  slightly.  The  bacteria  will  usually  appear 
more  distinct  if,  directly  after  pouring  off  the  stain,  the  prepara- 
tion is  rinsed  for  a  few  seconds  in  i  per  cent  solution  of  acetic 
acid,  and  then  thoroughly  washed  in  water.  The  acetic  acid 
solution  serves  to  remove  in  a  measure  any  color  which  has 
been  imparted  to  the  background,  and  which  is  undesirable. 

Preparations  that  are  mounted  at  first  in  water  may  be  made 
permanent  by  moistening  the  edge  of  the  cover-glass  so  that  it 
may  be  easily  removed  from  the  slide,  then  drying  and  mounting 
in  Canada  balsam.  Cover-glass  preparations  which  have  been 
stained  are  examined  with  the  oil-immersion  objective,  employ- 
ing the  plane  mirror,  having  the  iris  diaphragm  open  and  the 
condenser  close  to  the  lower  surface  of  the  glass  slide.  The 
purpose  is  to  obtain  the  most  intense  illumination  possible  over 
a  small  field. 

Gram's  Method. — Cover-glass  preparations,  having  been 
prepared  and  fixed  in  the  usual  manner  (see  page  38),  are  stained 
as  follows: 

(a)  Stain  in  aniline-water  gentian  violet  solution,  from  two 
to  five  minutes.  The  intensity  of  the  stain  may  be  increased 
by  warming  slightly. 


THE   MICROSCOPE   AND   MICROSCOPIC   METHODS  45 

(b)  Gram's  solution,  one  and  one-half  minutes: 

Iodine ,        i  gram. 

Potassium  iodide 2  grams. 

Water 300  c.c. 

In  this  solution  the  preparation  becomes  nearly  black. 

(c)  Wash  in  alcohol  repeatedly;  the  alcohol  becomes  stained 
with  clouds  of  violet  coloring  matter;  the  alcohol  is  used  as  long 
as  the  violet  color  continues  to  come  away,  and  until  the  prepara- 
tion is  decolorized  or  has  only  a  faint  steel-blue  color. 

(d)  When  desired,  the  specimens  may  be  stained,  by  way  of 
contrast,    with   a   watery   solution   of   Bismarck   brown,    dilute 
fuchsin  or  eosin. 

(e)  Wash  in  water,  and  examine  either  in  water  directly  or 
after  drying  and  mounting  in  Canada  balsam.     Gram's  method 
and  its  modifications  should  not  be  regarded  as  absolute  means 
of  distinguishing  between  Gram-positive  and  Gram-negative  bac- 
teria in  every  case,  as  much  depends  upon  the  condition  of  the 
bacteria,  and  very  much  upon  the  technic  of  staining.     When  the 
Gram  stain  is  used  for  diagnosis,  it  is  well  to  put  a  smear  of  a 
known  Gram-negative  and  a  smear  of  a  known  Gram-positive 
organism  on  the  same  slide  or  cover-glass  along  with  the  un- 
known, and  subject  them  all  to  the  same  technic. 

Some  bacteria  that  are  stained  by  Gram's  method : 
Staphylococcus  aureus, 
Streptococcus  pyogenes, 
Micrococcus  lanceolatus  (of  pneumonia), 
Micrococcus  tetragenus, 
Bacillus  of  diphtheria, 
Bacillus  of  tuberculosis, 
Bacillus  of  leprosy, 
Bacillus  of  anthrax, 
Bacillus  of  tetanus, 

Bacillus  welchii  (  aerogenes  capsulatus), 
Ray  fungus  of  actinomycosis. 


46  BACTERIOLOGY 

Of  these  the  tubercle  bacillus  and  the  bacillus  of  leprosy 
require  a  much  longer  exposure  to  the  stain  than  other  bacteria 
in  the  list. 

Some  bacteria  that  are  not  stained  by  Gram's  method : 
Gonococcus, 

Diplococcus  intracellularis  (meningitidis) , 
Micrococcus  melitensis, 
Bacillus  of  chancroids  (Ducrey), 
Bacillus  of  dysentery  (Shiga), 
Bacillus  of  typhoid  fever, 
Bacillus  coli, 
Bacillus  pyocyaneus, 
Bacillus  of  influenza, 
Bacillus  of  bubonic  plague, 
Bacillus  of  glanders  (Bacillus  mallei), 
Bacillus  proteus, 
Spirillum  of  Asiatic  cholera, 
Spirillum  of  relapsing  fever. 

Staining  of  Acid-proof  Bacteria. — A  very  large  number  of 
methods  have  been  proposed  for  staining  the  tubercle  bacillus, 
all  of  which  depend  upon  the  principle  that,  after  adding  to 
solutions  of  aniline  dyes  certain  substances,  like  aniline  water, 
carbolic  acid,  or  solutions  of  ammonia  or  soda,  the  tubercle  bacillus 
is  stained  with  great  intensity,  and  gives  up  its  stain  with  difficulty. 
Solutions  of  acids  will  remove  the  stain  from  all  parts  of  the  prepa- 
ration excepting  from  the  tubercle  bacilli,  which  retain  the  dye, 
having  once  acquired  it.  The  rest  of  the  preparation  may  now 
be  given  a  different  color  —  contrast-stain. 

Bacilli  that  resist  decolorization  by  acids  are  called  acid-proof 
or  acid-fast. 

Some  acid-proof  bacteria: 
Bact.  tuberculosis, 
Bact.  leprae, 
Bact.  smegmatis, 
Grass  bacillus  of  Moeller, 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS       47 

Butter  bacillus  of  Rabinowitsch, 

Certain  strep  to  thrices, 

Certain  bacilli  common  in  the  feces  of  cattle, 

Certain  bacteria  found  in  distilled  water, 

Spores  of  many  bacteria. 

Occasionally  other  bacteria,  micrococci  and  horny  epithelial 
cells  are  imperfectly  decolorized,  but  their  forms  distinguish 
them  from  tubercle  bacilli.  Minute  crystalline  needles  which 
have  a  shape  like  that  of  bacilli,  are  often  encountered  in  sputum, 
but  their  nature  will  be  recognized  after  a  little  practice. 

The  stain  for  acid-proof  bacteria  is  most  frequently  used  for 
specimens  of  sputum  from  cases  of  suspected  pulmonary  tubercu- 
losis; it  may  be  applied  to  other  fluids  and  secretions  equally 
well.  It  is  not  reliable,  however,  when  applied  to  milk,  as  the 
oil  present  in  milk  interferes  with  its  operation,  and  milk 
and  its  products  quite  often  contain  other  acid-proof  bacilli. 
The  smegma  of  the  external  genitals  also  frequently  contains 
acid-proof  bacilli  that  are  not  tubercle  bacilli.  On  this  account 
all  fluids  and  discharges  from  the  genito-urinary  tract  need  to 
be  examined  with  particular  care  not  to  confuse  tubercle  bacilli 
with  smegma  bacilli.  Too  much  reliance  should  not  be  placed 
on  the  possibility  of  distinguishing  between  tubercle  and  smegma 
bacilli  by  decolorizing  in  alcohol.  In  doubtful  cases  an  animal 
should  be  inoculated. 

Patients  should  be  given  minute  instructions  concerning  the 
collection  of  sputum.  The  bottle  used  should  be  new,  wide- 
mouthed,  clean,  and  kept  tightly  stoppered  with  a  clean  cork. 
The  patient  should  be  cautioned  against  allowing  the  expectora- 
tion to  get  on  the  outside  of  the  bottle.  Probably  whatever 
risk  is  incurred  by  those  who  examine  sputum  comes  chiefly 
from  the  outside  of  the  bottle  having  been  soiled  with  sputum 
containing  tubercle  bacilli.  It  is  well  to  disinfect  the  exterior 
of  the  bottle  when  it  is  received  at  the  laboratory.  Often  little 
white  particles  may  be  seen  floating  in  the  mucous  portions  of 
the  sputum.  These  particles  should  be  selected  for  the  investiga- 


48  BACTERIOLOGY 

tion,  and  may  be  spread  in  a  thin  film  on  the  cover-glass  with  the 
platinum  wire,  which  is  sterilized  in  the  flame  before  and  after 
using.  The  selection  of  the  little  white  particles  will  be  faciliated 
if  the  sputum  be  poured  into  a  clean  glass  dish,  which  may  be 
placed  on  a  black  surface.  A  form  of  porcelain  dish  is  furnished 
by  dealers,  the  bottom  of  which  is  black,  and  which  is  convenient 
for  these  manipulations.  The  smears  may  be  made  moderately 
thick  as  a  larger  amount  of  sputum  may  thus  be  examined  in 
a  short  time.  Uniform  thickness  is  difficult  to  obtain  and  is  not 
absolutely  essential.  It  is  hardly  necessary  to  observe  that  the 
operator  must  be  scrupulously  careful  not  to  contaminate  the  ma- 
terial under  examination  with  any  kind  of  extraneous  matter. 
The  cover-glasses  and  slides  which  are  used  should  be  new,  and 
should  have  been  cleaned  with  bichromate  of  potassium  and 
sulphuric  acid  (see  page  36).  When  the  work  is  completed,  the 
bottle  containing  the  sputum  should  be  sterilized  by  steam  or 
boiling. 

Method  for  staining  the  tubercle  bacillus: 

(a)  The  cover-glass  or  slide  preparation  is  made,  dried,  and 
fixed  by  passing  through  the  flame  three  times. 

(b)  The  cover-glass,  held  in  forceps  or  in  a  watch-crystal  is 
covered   with   steaming   carbol-fuchsin   for  five   minutes.     If   a 
slide  is  employed  it  may  be  conveniently  stained  in  the  Krauss 
staining  dish,  being  turned  face  downward. 

(c)  Wash  in  water. 

(d)  Wash  in  alcohol  containing  3  per   cent  of   hydrochloric 
acid  one  minute,  or  longer  if  necessary  to  remove  the  red  color. 

(e)  Wash  in  water. 

(f)  Stain  with  methylene-blue  solution  (see  page  40)  thirty 
seconds. 

(g)  Wash  in  water. 

(h)  Examine  in  water  directly,  and  after  drying  and  mounting 
in  Canada  balsam.  If  the  preparation  has  been  made  on  a  slide 
it  may  be  dried  and  examined  directly  in  cedar  oil  with  the  iV  in. 
objective.  When  the  preparation  is  mounted  in  water,  tubercle 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS       49 

bacilli  may  be  obscured  by  refraction  in  the  thicker  portions  of 
the  smear.     Tubercle  bacilli  take  a  brilliant  red  color;   other_ 
bacteria  and  the  nuclei  of  cells  are  stained  blue. 

Of  the  numerous  methods  of  staining  tubercle  bacilli  only 
a  few  others  can  be  mentioned.  Aniline- water  fuchsin,  aniline- 
water  gentian  violet,  or  carbol-fuchsin  may  be  used.  The  in- 
tensity of  the  stain  must  then  be  increased  by  warming  the  prepa- 
ration till  it  steams  or  boils,  then  allowing  the  warm  stain  to 
act  on  the  specimens  for  from  three  to  five  minutes;  the  prepara- 
tion may  also  be  left  in  the  cold  stain  over  night.  Decoloriza- 
tion  may  be  effected  with  a  25  per  cent  solution  of  sulphuric 
acid  used  till  the  red  color  disappears,  or  a  30  per  cent  solution 
of  nitric  acid,  which  operates  very  rapidly.  If  the  red  color 
persists  after  washing  in  water,  dip  in  the  acid  again.  After 
either  acid  the  preparation  is  to  be  washed  in  alcohol  until  the 
last  trace  of  the  stain  has  been  removed.  An  excellent  de- 
colorizing agent  is  a  3  per  cent  solution  of  hydrochloric  acid  in 
alcohol,  used  for  about  a  minute.  The  contrast  stain  may  be 
omitted  entirely  if  it  is  .desired.  A  suitable  contrast  stain  after 
fuchsin  staining  is  a  solution  of  methylene  blue;  after  gentian- 
violet  staining,  Bismarck  brown. 

Those  who  have  had  experience  in  staining  tubercle  bacilli 
soon  discover  that  the  bacilli  exhibit  some  differences  in  their 
resisting  power  to  strong  acids.  One  encounters  occasionally 
bacilli  that  are  perfectly  stained  side  by  side  with  others  that  are 
more  or  less  completely  decolorized.  These  facts  show  the 
necessity  of  practice  with  any  method,  and  of  exercising  caution 
and  judgment  in  making  a  diagnosis  where  the  number  of  bacilli 
happens  to  be  scanty.  If  tubercle  bacilli  are  not  found  in  the 
first  preparation,  other  preparations  should  be  made.  Some- 
times a  large  number  of  cover-glasses  must  be  examined. 

Various  expedients  have  been  devised  to  concentrate  tubercle 
bacilli  when  only  a  small  number  may  be  present  in  a  sample  of 
sputum.  Recently,  antiformin  (a  preparation  of  chlorinated 
sodium  hydroxide)  has  been  employed  for  this  purpose.  The  fol- 


50  BACTERIOLOGY 

lowing  method  is  that  of  Williamson.1  The  sputum  is  measured 
and  transferred  to  a  clean  flask  of  Jena  glass.  An  equal  volume  of 
50  per  cent  antiformin  is  added,  mixed  with  the  sputum,  and  the 
mixture  brought  to  a  boil  over  the  flame.  This  dissolves  the 
sputum  promptly.  The  material  is  then  cooled  and  to  each  10  c.c. 
of  material  in  the  flask  1.5  c.c.  of  a  mixture  of  chloroform,  one  part, 
and  alcohol,  nine  parts,  is  added.  The  mixture  is  thoroughly 
shaken.  As  a  result  the  tubercle  bacilli  imbibe  some  of  the  chloro- 
form and  become  heavier.  The  material  is  next  centrifugalized 
at  high  speed  for  15  minutes,  which  separates  it  into  three  layers, 
antiformin  above  and  chloroform  below  with  the  layer  of  sediment 
between  the  two.  This  layer  is  removed  and  mixed  with  egg 
albumen  (egg  albumen +0.5  per  cent  carbolic  acid)  on  a  slide 
and  then  spread  into  a  smear  between  two  slides.  The  smears 
are  then  dried  and  stained  in  the  usual  way.  Instead  of  using 
albumen  to  fix  the  sediment  to  the  slide,  it  is  convenient  to  save 
some  of  the  original  sputum  and  mix  it  with  the  sediment  for 
this  purpose. 

Staining  of  Spores. — The  method  is  applicable  to  cover- 
glass  preparations  which  may  be  prepared  in  the  usual  way  from 
material  supposed  to  contain  spores. 

(a)  After  drying  the  smear  on  the  cover-glass,  fix  it  with  heat 
by  passing  through  the  flame  three  times. 

(b)  Float  the  cover-glass  face  downward  on  the  surface  of 
steaming  hot  carbol-  fuchsin  or  aniline- water  fuchsin  for  three  to 
five  minutes. 

(c)  Wash  in  3  per  cent  hydrochloric  acid  alcohol  one  minute, 
or  less. 

(d)  Wash  in  water. 

(e)  Stain  with  watery  solution  of  methylene  blue  half  a  minute. 
(/)  Wash. 

(g)  Dry. 
(h)  Balsam. 

The  spores  are  intensely  stained  by  the  fuchsin.     The  stain 
1  Williamson,  Journ.  A.M.  A.,  Apr.  6,  1912,  Vol.  LVIII,  p.  1005-07. 


THE   MICROSCOPE   AND    MICROSCOPIC   METHODS  51 

is  removed  from  everything  except  the  spores  by  the  acid  alcohol. 
The  methylene-blue  solution  stains  the  bodies  of  the  bacteria, 
the  spores  remaining  brilliant  red.  There  are  various  other 
methods  for  staining  spores,  but  this  procedure  usually  gives 
good  results.  The  principle  is  the  same  as  in  staining  the  tubercle 
bacillus,  except  that  more  pains  are  needed  to  impregnate  spores 
with  the  dye. 

When  it  fails,  the  cover-glass  preparation  may  be  treated  by 
Moeller's  method  previous  to  staining.  After  fixation,  the  prep- 
aration is  immersed  in  chloroform  for  2  minutes,  drained  and 
dried  in  the  air.  It  is  then  immersed  in  5  per  cent  chromic 
acid  for  2  minutes,  washed  thoroughly  in  water,  and  stained 
as  above  described. 

Staining  of  Capsules. — The  capsules  which  many  bacteria 
possess,  appear  to  be  made  of  some  gelatinous  substance,  which 
is  difficult  to  stain. 

Method  of  Welch. — (a)  Cover-glass  preparations  are  made 
in  the  usual  manner.  Pour  glacial  acetic  acid  over  the  film. 

(&)  After  a  few  seconds,  replace  with  aniline-water  gentian 
violet,  without  washing  in  water.  Change  the  stain  several 
times  to  remove  all  the  acetic  acid.  Allow  it  to  act  three  or 
four  minutes. 

(c)  Wash  and  examine  in  sa,lt  solution  0.8  to  2.0  per  cent. 
Bacteria  are  deeply  stained,  while  their  capsules  are  pale  violet. 
This  method  has  been  recommended  for  staining   the  capsule 
of  the  pneumococcus. 

Methods  of  Hiss. — i.  (a)  Cover-glass  preparations  are  made 
in  the  usual  manner,  and  fixed  in  the  flame. 

(b)  Stain  for  a  few  seconds  in  a  half- saturated  watery  solu- 
tion of  gentian  violet. 

(c)  Wash  in  25  per  cent  solution  of  potassium  carbonate  in 
water. 

(d)  Mount  and  study  in  the  same. 

2.  (a)  Cover-glass  preparations  are  made  and  fixed  in  the 
ordinary  way. 


5  2  BACTERIOLOGY 

(b)  Use  the  following  stain,  heated  till  it  steams : 

Saturated  alcoholic  solution  of  gentian  violet  or  fuchsin 5  c.c. 

Distilled  water 95  c.c. 

(c)  Wash  in  20  per  cent  solution  of  cupric  sulphate  crystals. 

(d)  Dry  and  mount  in  Canada  balsam. 

The  methods  of  Hiss  are  recommended  to  be  used  for  bac- 
teria that  have  been  cultivated  on  serum-agar  with  i  per  cent  of 
dextrose.  They  have  shown  that  many  streptococci  have  cap- 
sules. The  writer  has  had  good  success  from  the  latter  method, 
with  preparations  of  the  pneumococcus  from  animal  tissues. 

Staining  of  Flagella. — Flagella  are  among  the  most  difficult 
of  all  objects  to  stain.  The  best-known  method  is  that  of 
Loffler.  It  is  important  to  use  young  cultures  (4  to  10  hours 
old),  preferably  on  agar. 

(a)  A  small  amount  of  the  growth  is  gently  mixed  with  a 
large  drop  of  distilled  water  on  a  clean  slide,  so  that  the  water  is 
made  very  faintly  cloudy.     From  the  top  of  this  drop  one  or 
two  transfers  are  made  to  a  second  drop  with  a  small  platinum 
loop.     From  this  second  drop  a  loopful  is  transferred  to  a  per- 
fectly clean  (flamed)  cover-glass,  spread  with  minimum  manipu- 
lation and  dried  quickly,  high  over  the  flame. 

(b)  After  drying,  fixation  is  effected  by  passing  through  the 
flame  three  times,  holding  the  cover-slip  between  the  thumb  and 
fore  finger  to  avoid  overheating. 

(c)  The  essential  point  in  this  method  is  the  use  of  a  mordant 
as  follows : 

Tannic  acid,  10  per  cent  solution  20  c.c. 

Saturated  solution  of  ferrous  sulphate 4  c.c. 

Saturated  alcoholic  solution  of  fuchsin i  c.c. 

This  solution  should  be  freshly  prepared  from  pure  substances, 
and  should  be  filtered  at  once  after  mixing.  It  may  deteriorate 
in  a  few  hours  but  sometimes  keeps  for  a  few  days  or  weeks. 
A  few  drops  are  placed  on  the  cover-glass,  or  the  cover-glass  is 


THE   MICROSCOPE   AND   MICROSCOPIC  METHODS  53 

placed,  face  down,  in  a  dish  containing  the  stain;  it  is  then  left 
for  one  to  five  minutes,  warming  slightly. 

(d)  Wash  in  water. 

(e)  Stain  with  aniline-water  fuchsin,  or  carbol-fuchsin. 
(/)  Wash  in  water. 

(g)  Dry. 

(ti)  Mount  in  Canada  balsam.    , 

(According  to  Loffler,  certain  bacteria  require  the  addition  of 
an  acid  solution,  and  certain  others  an  alkaline  solution,  but  many 
observers  consider  this  unnecessary.) 

Another  and  very  valuable  method  is  that  of  Van  Ermen- 


(a)  Make  and  fix  cover-glass  preparations  as  in  the  preceding 
method. 

(b)  Use  the  following  mordant  for  one-half  hour    at    room 
temperature  or  for  five  minutes  at  50°  to  60°  C. 

Osmic  acid  2  per  cent  solution i 

Tannic  acid  10  to  25  per  cent  solution 2 

(c)  Wash  carefully  in  distilled  water  and  then  in  alcohol. 

(d)  Place  for  a  few  seconds  in  a  0.25  to  0.50  per  cent  solution  of 
nitrate  of  silver — "the  sensitizing  bath." 

(e)  Without  washing  transfer  to  the  "reducing  and  reinforcing 
bath": 

Gallic  acid 5  grams. 

Tannic  acid 3  grams. 

Fused  potassium  acetate 10  grams. 

Distilled  water 350  c.c. 

(/)  After  a  few  seconds,  replace  the  preparation  in  the  nitrate 
of  silver  solution,  in  which  it  is  kept  constantly  moving,  till  the 
solution  begins  to  acquire  a  brown  or  black  color.  Some  recom- 
mend leaving  the  preparation  in  the  nitrate  of  silver  solution  for 
two  minutes  in  the  first  place,  and  in  the  reducing  bath  for  two 
minutes,  without  using  the  nitrate  of  silver  solution  a  second  time. 


54  BACTERIOLOGY 

(g)   Finally  wash  in  distilled  water,  dry,  mount  in  Canada 
balsam.     It  is  difficult  to  avoid  the  formation  of  precipitates; 
otherwise  the  results  of  this  method  are  usually  good. 

Wet  Fixation  of  Protozoa. — The  fluid  containing  the  protozoa 
is  spread  on  a  cover-glass  or  slide  and  immediately  dropped  upon 
a  solution  of  the  fixing  agent,  commonly  sublimate  alcohol  heated 
to  60°  C.  This  is  prepared  by  mixing  saturated  aqueous  solu- 
tion of  mercuric  chloride,  100  c.c.,  with  absolute  alcohol, 
50  c.c.,  and  acetic  acid,  5  drops.  After  a  few  minutes  the  prepa- 
ration is  carefully  washed  in  water,  and  passed  through  graded 
alcohols  to  harden.  It  may  then  be  stained,  dehydrated  in 
graded  alcohols,  cleared  in  xylol  and  mounted  in  balsam.  The 
preparation  should  not  be  allowed  to  dry  at  any  stage  of  the 
process. 

Haidenhain's  Iron  Hematoxylin. — The  preparation  to  be 
stained  by  this  method  should  be  fixed  in  mercuric  chloride  or 
alcohol.  The  stain  is  prepared  by  dissolving  hematoxylin  crys- 
tals, i  gram,  in  hot  absolute  alcohol  10  c.c.,  and  then  adding  dis- 
tilled water  90  c.c.  This  solution  is  allowed  to  stand  in  an  open, 
cotton-plugged  bottle  for  about  four  weeks,  and  it  is  then  diluted 
with  an  equal  volume  of  water  before  using.  The  iron  solution 
is  made  by  dissolving  2.5  grams  of  ferric  ammonium  sulphate 
(lavender-colored  crystals)  in  100  c.c.  of  distilled  water.  The 
preparation  to  be  stained  is  first  soaked  in  the  iron  solution 
for  four  to  eight  hours,  then  rinsed  and  immersed  in  the  hema- 
toxylin for  twelve  to  twenty-four  hours.  It  is  again  rinsed  and 
now  differentiated  by  immersion  in  the  iron  solution  until  black 
clouds  cease  to  be  given  off.  When  the  desired  differentia- 
tion has  been  obtained  the  preparation  is  washed,  dehydrated 
by  passing  through  graded  alcohols,  and  absolute  alcohol,  cleared 
in  xylol  and  mounted  in  balsam. 

Preparation  and  Staining  of  Blood  Films. — Blood  films  are 
best  made  on  clean,  flamed  slides.  A  small  drop  of  fresh  blood  is 
received  on  the  surface  of  one  slide  near  one  end.  The  end  of 
another  slide  is  applied  to  the  first  at  an  acute  angle  so  that  the 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        55 

blood  spreads  laterally  in  the  angle  between  the  two  slides. 
The  second  slide  is  then  pushed  along  the  surface  of  the  first  with 
the  blood  following  it  in  the  angle.  The  thickness  of  film  may 
be  regulated  by  varying  the  size  of  angle  between  the  two  slides. 

For  staining  blood  films,  either  Leishman's  or  Giemsa's  stain 
or  some  modification  of  them  should  be  used  as  a  general  rule. 
After  fixation  in  absolute  alcohol,  blood  films  may  be  stained 
with  Loffler's  methylene-blue  or  by  Gram's  method. 

Staining  Bacteria  in  Tissues. — Pieces  of  organs  about  i  cm. 
in  thickness  may  be  taken.  Alcohol  is  the  best  agent  for  pre- 
serving them.  The  hardening  will  be  completed  in  a  few  days. 
It  is  best  to  change  the  alcohol.  The  amount  of  the  alcohol  must 
be  twenty  times  the  bulk  of  the  tissue  to  be  preserved. 

Ten  parts  of  the  standard  40  per  cent  solution  of  formalde- 
hyde, with  90  parts  water  make  a  good  mixture  for  fixation;  after 
twenty-four  hours  change  to  alcohol. 

Imbedding  in  Collodion  or  Celloidin. — From  alcohol  the 
pieces  of  tissue  are  placed  in  equal  parts  of  alcohol  and  ether 
twenty-four  hours;  thin  collodion  (ij  per  cent),  twenty-four 
hours;  thick  collodion  of  a  syrupy  consistency  (6  per  cent)  twenty- 
four  hours.  The  specimen  is  laid  upon  a  block  of  wood  and  sur- 
rounded by  thick  collodion,  and  then  inverted  in  70  per  cent  al- 
cohol. The  collodion  makes  a  firm  mass,  surrounding  and  perme- 
ating the  tissue,  and  permits  very  thin  sections  to  be  cut.  The 
soluble  cotton  sold  by  dealers  in  photographer's  supplies  serves 
as  well  as  the  expensive  preparation  known  as  celloidin.  To 
make  collodion,  dissolve  it  in  equal  parts  of  alcohol  and  ether. 
Soluble  cotton  is  also  called  pyroxylin,  and  is  a  kind  of  gun-cotton. 

Imbedding  in  Paraffin. — (a)  Pieces  of  tissue  2  to  3  mm. 
thick  which  have  already  been  fixed  in  alcohol  or  formaldehyde 
are  to  be  placed  in  absolute  alcohol  for  twenty-four  hours. 

(b)  In  pure  xylol  one  to  three  hours. 

(c)  In  a  saturated  solution  of  paraffin  in  xylol  one  to   three 
hours. 

(d)  In   melted   paraffin  having   a   melting-point   of    50°    C., 


56  BACTERIOLOGY 

which  requires  the  use  of  a  water-bath  or  oven,  one  to  three 
hours.  The  xylol  must  be  entirely  driven  off,  and  the  tissue 
thoroughly  infiltrated. 

(e)  Change  to  fresh  paraffin  for  one  hour. 

(/)  Finally,  place  the  tissue  in  a  small  dish  or  paper  box  and 
pour  the  melted  paraffin  about  it.  Harden  as  quickly  as  possible 
with  running  water.  It  is  important  to  fix  the  piece  of  tissue 
in  a  suitable  position,  if  the  position  is  of  importance,  before 
pouring  in  the  melted  paraffin.  Sections  of  exquisite  thinness 
may  now  be  cut.  The  knife  need  not  be  wet.  Paraffin  im- 
bedding is  especially  desirable  when  serial  sections  are  to  be  made. 

In  order  to  mount  the  sections,  proceed  as  follows: 

(a)  Place  the  sections  on  water  in  a  porcelain  capsule. 
Warm  slightly,  when  the  sections  will  flatten  nicely.     Smear  the 
surface  of  a  slide  with  a  very  thin  layer  of  Mayer's  glycerin- 
albumen  mixture.     Dip  the  slide  under  the  sections;  lift  them; 
and  then  drain  off  the  water,  leaving  the  sections  in  their  proper 
positions.     Let  them  dry  for  some  hours  in  the  incubator,  and 
they  will  be  firmly  fastened  to  the  slide. 

GLYCERIN- ALBUMEN   MIXTURE    (MAYER). 

Equal  parts  of  white  of  egg  and  glycerin  are  thoroughly  mixed,  and  then 
filtered.  Add  a  little  gum-camphor  to  preserve. 

(b)  Dissolve  out  the  paraffin  in  one  of  the  numerous  solvents 
(xylol,  a  few  minutes) . 

(c)  At  this  point  the  xylol  should  be  washed  off  with  absolute 
alcohol,  and  then  70  per  cent  alcohol  and  finally  distilled  water. 

(d)  The  section  is  stained. 

(e)  Dehydrate  in  absolute  alcohol. 
(/)  Clear  in  xylol. 

(g)  Mount  in  balsam. 

Section  Cutting. — Cutting  is  best  done  with  an  instrument 
called  a  microtome.  The  tissues  may  be  imbedded  in  collodion 
or  paraffin;  or  when  they  have  been  hardened  with  formaldehyde 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        57 

they  may  be  cut  after  freezing.  Bacteria  stain  admirably  in 
frozen  sections.  For  routine  work  collodion  imbedding  will  be 
found  as  convenient  a  process  as  any.  Paraffin  imbedding  gives 
the  thinnest  sections. 

A  microtome  consists  of  a  heavy,  sliding  knife-carrier,  which 
moves  with  great  precision  on  a  level,  and  of  a  device  for  elevating 
the  object  which  is  to  be  cut,  any  desired  distance  after  each  ex- 
cursion of  the  knife.  The  thickness  of  the  section  will  be  the 


FIG.  27. — Schanze  microtome. 

distance  which  the  object  is  elevated.  The  knife  is  kept  wet 
with  alcohol  during  the  cutting  of  collodion  sections,  otherwise 
it  is  left  dry.  The  microtome  is  usually  provided  with  a  special 
form  of  knife.  A  razor  will  serve  nearly  as  well,  after  having 
had  the  lower  side  ground  flat.  If  a  razor  is  used,  a  special  form 
of  razor-holder  must  be  attached  to  the  microtome  to  receive  the 
razor.  Above  all,  it  is  necessary  that  the  knives  should  be  kept 
in  good  condition.  Only  occasionally  will  they  need  honing,  using 
a  fine  water-stone  or  Belgian  hone.  The  movement  in  honing 


58  BACTERIOLOGY 

should  be  from  heel  to  toe,  always  placing  the  back  of  the  knife 
next  the  hone  when  turning.  The  knife  should  be  stropped  fre- 
quently. The  leather  of  the  strop  should  be  glued  to  a  strip  of 
wood  to  make  a  flat  surface.  The  movement  in  stropping  should 
be  from  toe  to  heel.  Sections  should  be  cut  to  a  thickness  of  not 
more  than  25  /*.  Thinner  sections  (5  to  10  //)  are  to  be  desired. 

Staining  of  Sections. — A  watery  solution  of  one  of  the  aniline 
dyes  is  used — fuchsin,  gentian  violet  or  methylene  blue — made 
by  adding  a  few  drops  of  the  alcoholic  solution  to  a  dish  filled 
with  water.  Loffler's  solution  of  methylene  blue  serves  very 
well. 

By  this  process  most  bacteria  are  stained;  also  the  nuclei  of 
cells;  frequently,  also,  certain  granules  contained  within  some 
cells  (German,  Mastzellen),  which  may  easily  be  mistaken  for 
bacteria  by  the  inexperienced  (basophilic  granules). 

(a)  Place  the  section  in  the  staining  solution  from  two  to  five 
minutes. 

(b)  Wash  in  water. 

(c)  Place  in  a  watery  solution  of  acetic  acid,  i   per  cent  for 
one  minute. 

(d)  Alcohol,  one  to  two  minutes;  change  to  absolute  alcohol. 
Touch  the  sections  to  blotting-paper  to  remove  the  superfluous 
alcohol. 

(e)  Xylol  until  clear;  xylol  is  to  be  preferred  to  other  clearing 
agents,  like  oil  of  cloves,  most  of  which  slowly  remove  aniline 
colors.     It  has  the  disadvantage  of  not  clearing  when  more  than  a 
trace  of  water  is  present;  dehydration  in  alcohol  must,  therefore, 
be  complete.     The  section  should  be  removed  from  the  xylol  as 
soon  as  it  is  cleared;  otherwise  wrinkling  occurs. 

(/)  The  section  is  placed  upon  a  glass  slide;  a  drop  Canada 
balsam  is  placed  upon  it  and  then  a  cover-glass.  The  Canada 
balsam  should  be  dissolved  in  xylol. 

The  section  is  to  be  manipulated  with  straight  or  bent  needles. 
The  removal  from  xylol  to  the  glass  slide  is  managed  best  with  a 
spatula  or  section-lifter. 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        59 

The  above  statements  apply  to  frozen  sections  or  to  sections 
imbedded  in  celloidin.  Paraffin  sections  are  preferably  attached 
to  the  slide  with  glycerin-albumen.  The  different  steps  in  the 
process  follow  in  the  same  order.  The  stain  may  be  poured  on 
the  slide,  or  the  slide  may  be  placed  in  a  large  dish  full  of  staining 
fluid.  (See  page  44.)  Celloidin  sections  may  also  be  stained  on 
the  slide.  If  the  section  be  well  spread  and  flattened  thoroughly 
with  blotting  paper,  it  will  usually  adhere  to  the  slide,  and  is  less 
likely  to  wrinkle.  It  must  not  be  allowed  to  dry. 

Gram's  Method  may  be  applied  to  the  staining  of  sections 
of  tissues  as  well  as  to  smears  upon  cover-glasses. 

(a)  Place  the  section  in  aniline-water  gentian  violet,  one  to 
five  minutes. 

(b)  Rinse  briefly  in  water.- 

(c)  Iodine  solution  (see  page  45),  one  and  one-half  minutes. 

(d)  Alcohol,  until  decolorized  to  a  faint  blue-gray. 

(e)  Xylol. 

(/)  Mount  on  a  slide  in  balsam. 

Weigert's  Modification  of  Gram's  Method,  or  Weigerfs 
Stain  for  Fibrin. — (a)  Place  the  section  in  aniline-water  gentian 
violet  solution,  five  minutes  or  more. 

(b)  Wash  briefly  in  water. 

(c)  Place  the  section  upon  a  slide  by  means  of  a  section  lifter; 
having  straightened  it  carefully,  absorb  the  water  with  blotting- 
paper. 

(d)  Gram's  solution  (see  page  45)  one  to  two  minutes. 

(e)  Absorb  the  iodine  solution  with  blotting-paper. 

(/)  Add  aniline  oil,  removing  it  from  time  to  time  with  blotting- 
paper,  and  adding  fresh  aniline  oil  until  the  color  ceases  to  come 
away.  (Aniline  oil  serves  in  this  connection  both  to  decolorize 
and  to  dehydrate.  It  absorbs  the  water  rapidly  and  efficiently. 
However,  on  account  of  its  decolorizing  tendency,  it  must  be  re- 
moved before  the  specimens  can  be  mounted  permanently.) 

(g)  Add  xylol;  remove  it  with  blotting-paper;  and  add  fresh 
xylol  several  times,  in  order  to  extract  the  last  trace  of  aniline  oil. 


60  BACTERIOLOGY 

(ti)  Mount  in  Canada  balsam. 

This  method  is  more  convenient  for  the  staining  of  sections 
than  the  Gram  method.  The  results,  however,  are  essentially 
the  same  as  far  as  the  bacteria  are  concerned;  fibrin  and  hyaline 
material  are  stained  blue,  bacteria  violet.  It  is  often  impossible 
to  decolorize  the  nuclei  completely  without  decolorizing  the 
bacteria  also.  The  parts  of  the  nuclei  which  remain  stained 
often  present  pictures  that  resemble  bacteria,  and  which  may 
lead  to  error  if  not  recognized.  Basophilic  granules  also  retain 
the  stain,  as  do  the  horny  cells  of  the  epidermis.  These  remarks 
apply  also  to  Gram's  method,  except  as  regards  fibrin.  Very 
beautiful  preparations  can  be  obtained  according  to  this  or  the 
Gram  method  when  the  sections  have  previously  been  stained 
in  carmine;  the  nuclei  will  then  be  colored  red,  bacteria  violet. 

Tubercle  bacilli  may  be  stained  in  sections  as  follows: 

(a)  Use  carbol-fuchsin,   or    aniline- water  gentian  violet    for 
one-half  to  two  hours  with  very  gentle  warming,  or  over  night 
without  warming. 

(b)  Wash  in  water. 

(c)  Decolorize  with  some  one  of  the  decolorizing  agents  men- 
tioned in  connection  with  the  staining  of  tubercle  bacilli  in  cover- 
glass  preparations,  preferably  3   per   cent  hydrochloric-acid   al- 
cohol.    Decolorization  must  be  continued  until  the  red  color 
has  disappeared,  which  requires  one-half  to  several  minutes. 

(d)  Wash  in  alcohol. 

(e)  Wash  in  water. 

(/)  Use  hematoxylin  as  a  contrast-stain  for  fuchsin  prepara- 
tions, and  carmine  for  gentian  violet  preparations,  (it  is  bet- 
ter to  stain  with  carmine  first  of  all  and  before  staining  the 
bacilli.  The  carmine  is  not  affected  by  the  subsequent 
treatment.) 

(g)  Wash  in  water. 

(h)  Alcohol. 

(*)  XyloL 

(/)  Balsam, 


THE  MICROSCOPE  AND  MICROSCOPIC  METHODS        6 1 

Nuclear  stains,  which  may  be  used  as  contrast-stains  for 
sections: 

DELAFIELD'S  HEMATOXYLIN. 

Hematoxylin  crystals 4  grams. 

Alcohol 25  c.c. 

Ammonia  alum 50  grams. 

Water 400  c.c. 

Glycerin 100  c.c. 

Methyl  alcohol , 100  c.c. 

Dissolve  the  hematoxylin  in  the  alcohol,  and  the  ammonia 
alum  in  the  water.  Mix  the  two  solutions.  Let  the  mixture 
stand  four  or  five  days  uncovered;  it  should  have  become  a  deep 
purple.  Filter  and  add  the  glycerin  and  the  methyl  alcohol. 
After  it  has  become  dark  enough,  filter  again.  Keep  it  a  month 
or  longer  before  using;  the  solution  improves  with  age.  At  the 
time  of  using,  filter  and  dilute  with  water  as  desired. 

LITHIUM-CARMINE  (ORTH). 

Carmine 2.5  grams. 

Saturated  watery  solution  of  lithium  carbonate.  100.0  c.c. 

Add  a  few  crystals  of  thymol.  The  carmine  dissolves  readily 
in  the  lithium  carbonate  solution.  Filter  the  stain  at  the  time  of 
using.  Sections  are  to  be  left  in  the  stain  five  to  twenty  minutes. 

Sections  stained  in  carmine  are  placed  directly  in  acid  alco- 
hol (i  part  hydrochloric  acid,  100  parts  70  per  cent  alcohol)  for 
five  to  ten  minutes.  They  acquire  a  brilliant  scarlet  color. 
When  used  as  a  contrast-stain  for  tissues  containing  bacteria, 
it  is  best  to  use  it  before  staining  the  bacteria,  which  might  be 
decolorized  by  the  acid  alcohol. 


CHAPTER  II. 

STERILIZATION— DISINFECTION— ANTISEPSIS- 
FOOD  PRESERVATION. 

Definitions. — By  sterilization  is  meant  the  killing  or  the  re- 
moval of  all  micro-organisms  in  or  on  a  body  or  substance. 
Disinfection  has  a  somewhat  analogous  signification,  but  denotes 
the  destruction  or  removal  of  infectious  microbes,  and  this  may 
or  may  not  be  accomplished  without  complete  sterilization, 
according  to  the  nature  of  the  particular  case  in  hand.  Anti- 
sepsis means  the  inhibition  of  growth  of  micro-organisms  with- 
out ordinarily  killing  or  removing  them,  and  is  especially  applied 
to  the  checking  of  microbic  activity  in  wounds  and  the  effects 
produced  thereby  (sepsis).  Food  preservation  involves  similar 
principles,  depending  upon  the  prevention  of  microbic  activity 
in  dead  organic  matter  either  by  sterilization  or  by  the  presence 
of  inhibitive  substances,  similar  to  antiseptics,  but  in  this  instance 
called  preservatives. 

In  connection  with  sterilization  we  shall  consider  those  agents 
which  remove  or  destroy  a  part  of  the  microbic  flora  without 
producing  complete  sterility,  as  well  as  the  methods  which  insure 
complete  sterilization.  A  few  examples  of  each  general  class 
will  be  considered. 

Physical  Sterilization. — Among  the  physical  means  by  which 
sterilization  may  be  accomplished,  those  which  are  merely  me- 
chanical may  be  mentioned  first.  The  removal  of  microbes  from 
an  infected  surface  by  washing  them  away  is  a  method  of  wide 
application.  Complete  sterility  may  sometimes  be  attained  in 
this  way.  In  ordinary  disinfection  of  woodwork,  walls  and 
floors,  or  of  the  hands,  mechanical  cleaning  is  of  primary  impor- 
tance, even  though  it  does  not  insure  complete  sterilization. 

62 


STERILIZATION — ANTISEPSIS — FOOD    PRESERVATION  63 

The  process  removes  not  only  many  of  the  bacteria,  but  also 
much  other  material  which  serves  to  protect  them  and  even  to_ 
furnish  food  for  their  development.  Another  mechanical  method 
is  that  of  comminution,  actual  crushing  of  the  bacterial  cells. 
It  is  of  very  narrow  application  and  not  to  be  relied  upon. 
High  pressures  have  been  employed  to  destroy  bacteria,  but 
hydrostatic  pressure  of  even  1000  atmospheres  does  not  produce 
complete  sterilization.  Sedimentation  is  a  method  of  primary 
importance,  especially  in  the  removal  of  suspended  bacteria  from 
the  atmosphere.  It  also  operates  to  remove  a  large  proportion 
of  the  bacteria  from  drinking  water  when  stored  in  suitable  reser- 
voirs. Filtration  of  fluids  is  an  important  means  of  sterilizing 
them.  Air  may  be  sterilized  by  drawing -it  slowly  through  a 
sufficient  layer  of  cotton.  Water  becomes  bacteria-free  as  it 
niters  through  the  soil,  so  that  waters  from  the  depths  of  the  earth 
are  sterile.  Liquids  are  commonly  sterilized  in  the  laboratory 
by  forcing  them  through  a  layer  of  unglazed  porcelain  (Pasteur- 
Chamberland  filter)  or  through  a  compact  wall  of  diatomaceous 
earth  (Berkefeld  filter).  Liquids  rich  in  bacteria,  such  for  ex- 
ample as  cultures  in  broth,  may  be  rendered  bacteria-free  in  this 
way.  These  filters  have  also  been  employed  for  the  sterilization 
of  drinking  water,  but  their  use  for  this  purpose  requires  intelli- 
gence and  care,  and  when  carelessly  employed  they  are  worse 
than  useless. 

Dessication  is  destructive  to  many  microbes,  especially  those 
which  do  not  form  spores.  The  germs  of  Asiatic  cholera  are  dead 
in  a  few  hours  after  complete  drying.  The  spores  of  the  anthrax 
bacillus  on  the  other  hand  remain  alive  for  at  least  ten  years  after 
drying.  Most  bacteria  resist  drying  long  enough  so  that  they 
may  be  transferred  by  air  currents  as  dust  and  still  be  capable  of 
growth. 

Light  is  injurious  to  bacteria  and  direct  sunlight  is  rapidly 
fatal  to  them,  even  in  spore  form.  Light  seems  to  act  by  pro- 
ducing powerful  chemical  germicides,  probably  organic  peroxides, 
in  the  medium  surrounding  the  bacteria.  Such  substances  are 


64  BACTERIOLOGY 

known  to  be  produced  under  these  circumstances.     They  rapidly 
decompose. 

Cold  appears  to  be  fatal  to  some  pathogenic  forms,  and  a  con- 
siderable percentage  of  the  bacteria  in  a  culture  are  usually  killed 
by  freezing.  Cultures  cannot  be  completely  sterilized  even  by 
exposure  to  the  temperature  of  liquid  air.  Cold  is  therefore  not 
to  be  regarded  as  an  efficient  germicide,  although  it  may  com- 
pletely check  the  growth  of  bacteria. 

Heat  is  the  most  important  of  the  physical  means  and 
doubtless  the  most  important  of  all  means  of  destroying 
bacteria.  Its  value  as  a  purifying  agent  was  recognized  among 
the  ancients.  Heat  is  applied  under  conditions  insuring 
the  presence  of  liquid  water,  so-called  moist  heat,  and  in 
the  absence  of  water,  so-called  dry  heat  or  hot- air  sterilization. 
The  most  reliable  methods  of  sterilization  by  dry  heat  are  those 
which  accomplish  the  combustion  or  destructive  distillation  of 
organic  matter  in  general.  Actual  combustion  of  clothing  and 
bedding,  and  even  of  houses  has  been  resorted  to  in  the  past 
as  a  method  of  disinfection.  Heating  to  redness  in  the  naked 
flame  is  the  routine  method  of  sterilizing  our  platinum  wire,  and 
glass  articles,  such  as  capillary  pipettes,  cover-glasses  and  slides 
are  commonly  sterilized  in  the  flame.  Flaming  may  even  be 
employed  for  sterilization  of  surgical  instruments  in  an  emergency, 
although  such  treatment  quickly  destroys  steel  instruments. 
Sterilization  of  large  objects  and  of  combustible  material  by  dry 
heat  is  generally  accomplished  in  an  oven  or  hot-air  sterilizer. 
The  common  laboratory  sterilizers  are  boxes  of  sheet  iron  with 
double  walls,  with  air  space  between  to  allow  the  hot  gases  from 
the  flame  completely  to  surround  the  inner  compartment.  The 
door,  which  occupies  one  full  side,  is  usually  double.  A  tubula- 
tion  through  the  top  allows  a  thermometer  to  be  inserted  into  the 
interior  so  that  the  temperature  may  be  read  off  at  any  time. 
Even  the  best  hot-air  sterilizers  fail  to  give  an  even  temperature 
all  over  the  interior,  so  that  the  thermometer  bulb  at  one  corner 
cannot  be  implicitly  relied  upon  to  record  the  temperature  of 


STERILIZATION — ANTISEPSIS — FOOD   PRESERVATION  65 

other  parts.  Ordinarily  a  temperature  of  150°  C.  for  one  hour, 
170°  C.  for  30  minutes,  or  200°  C.  for  one  minute  will  kill  all 
bacteria.  Such  exposure  browns  cotton  of  good  grade  only 
slightly.  One  fallacy  in  hot-air  sterilization  needs  to  be  guarded 
•against.  Glassware  and  other  apparatus  must  be  dry  before  it 
is  put  into  the  oven  to  sterilize.  A  tube  containing  water  may 
be  left  in  the  oven  until  the  thermometer  records  a  temperature 


FIG.  28. — Hot-air  sterilizer. 

of  200°  C.  in  the  upper  corner  of  the  sterilizer,  and  subsequently 
the  tube  may  be  removed  from  the  oven  with  the  most  of  the 
water  still  in  it.  Hot-air  sterilization  is  employed  for  glassware, 
tubes  with  cotton  plugs,  granite-ware,  stone-ware,  and  for  metals 
not  injured  by  heat. 

Moist  heat  or  heat  in  the  presence  of  liquid  water  must  be  used 
whenever  drying  is  to  be  avoided,  especially  in  the  sterilization  of 
culture  media  and  various  solutions.  It  is  employed  as  continu- 
ous sterilization  at  a  single  exposure  and  as  discontinuous  ster- 

5 


66 


BACTERIOLOGY 


ilization,  heating  for  a  short  time  on  several  consecutive  days. 
The  temperature  employed  varies  according  to  the  effect  desired. 
A  temperature  of  60°  C.,  maintained  throughout  a  watery  liquid 
for  twenty  minutes  will  kill  most  vegetative  bacteria,  and  practi- 
cally all  pathogenic  bacteria  which  do  not  form  spores.  Such 
partial  sterilization  is  called  Pasteurization.  Boiling  water,  100° 
C.,  kills  vegetative  bacteria  in  a  very  short  time,  less  than  two  min- 
utes for  most  bacteria,  and  the 
spores  of  many  species  are  de- 
stroyed by  boiling  for  5  to  30  min- 
utes. Some  spores,  however,  for 
example  those  of  some  varieties  of 
B.  vulgatus,  may  survive  a  boiling 
temperature  for  several  hours. 
Boiling  is  one  of  the  most  useful 
practical  methods  of  disinfection. 
Nearly  all  pathogenic  bacteria  are 
quickly  killed  in  boiling  water. 
Surgical  instruments  are  com- 
monly boiled  in  water  to  which 
sodium  carbonate,  i  to  2  per  cent, 
has  been  added.  Rusting  and 
corrosion  may  also  be  prevented 
by  adding  10  per  cent  of  borax 
to  the  water  in  which  metal  in- 
struments are  boiled.  Steriliza- 
tion of  bacteriological  media  is 

usually  done  by  means  of  streaming  steam,  rather  than  by  immer- 
sion in  boiling  water.  The  Koch  steam  sterilizer  is  a  compara- 
tively simple  device  for  this  kind  of  sterilization.  It  is  a  tall, 
cylindrical,  tin  vessel  covered  with  asbestos  or  felt.  The  lower 
portion  is  filled  with  water;  on  the  side  is  a  water-gauge  indicat- 
ing the  height  of  the  water,  in  order  .that  one  may  observe  when 
there  is  danger  of  the  sterilizer  boiling  dry.  Over  the  top  there 
is  a  tight-fitting  cover.  The  steam  is  generated  by  a  Bunsen 


FIG.  29. — Koch's  steam  sterilizer. 


STERILIZATION — ANTISEPSIS — FOOD   PRESERVATION 


67 


burner  standing  underneath.  A  perforated  shelf  placed  some  dis- 
tance above  the  surface  of  the  water  is  for  the  reception  of  the- 
tubes  and  flask  that  are  to  be  sterilized.  The  Arnold  steam  sterili- 
zation is  somewhat  more  complicated  but  is  very  convenient  and 
efficient.  It  consists  of  a  cylinder  of  tin  or  copper  with  a  cover, 
which  is  enclosed  in  a  movable  cylindrical  outer  cover  or  hood. 
The  inner  cylinder  has  an  opening  in  the  bottom  through  which 
steam  may  enter,  the  steam  com- 
ing from  a  small  chamber  under- 
neath with  a  copper  bottom  to 
which  the  flame  is  applied.  The 
peculiarity  of  this  form  of  steril- 
izer consists  in  the  fact  that  the 
steam  which  escapes  from  the 
sterilizing  chamber  condenses  be- 
neath the  outer  cover  or  hood  and 
falls  back  upon  the  pan  over  the 
chamber  in  which  the  steam  is 
generated.  The  bottom  of  this 
pan  is  perforated  with  three  small 
holes,  which  allow  the  water  of  con- 
densation to  return  into  the  cham- 
ber where  the  steam  is  generated. 
The  sterilizer,  therefore,  to  a  cer- 
extent,  supplies  itself  with 


tain 


FIG. 


30.— Diagram    of    the    Arnold 
steam  sterilizer. 


water,  although  not  by  any  means 
perfectly.  It  is,  however,  less 
likely  to  boil  dry  than  other  forms  of  sterilizers,  and  it  has  the 
advantage  of  being  reasonably  cheap  and  quite  effective.  The 
space  inclosed  by  the  hood  also  serves  as  a  steam-jacket  and  helps 
to  prevent  fluctuations  in  temperature.  A  great  improvement 
upon  the  ordinary  Arnold  sterilizer  is  the  modification  of  it  devised 
by  the  Massachusetts  Board  of  Health. 

In  the  use  of  this,  or  any  form  of  steam  sterilizer,  the  time  is 
noted  from  the  period  when  boiling  is  brisk  and  it  is  evident  that 


68 


BACTERIOLOGY 


the  sterilizing  chamber  is  filled  with  hot  steam;  or,  what  is  better, 
when  the  thermometer  registers  100°  C.,  if  the  sterilizer  be  pro- 
vided with  a  thermometer.  With  a  large  Arnold  sterilizer  a 
temperature  of  100°  C.  may  not  be  reached  intil  it  has  been 
heated  with  a  rose-burner  for  twenty  to  thirty-five  minutes. 
When  bulky  articles  or  large  amounts  of  material  are  to  be  ster- 
ilized, allowance  must  be  made  for  the  time  necessary  to  bring  the 
temperature  in  the  middle  of  the  mass  to  100°  C. 


FIG.  31. — Steam  sterilizer,  Massachusetts  Board  of  Health. 

Autoclave  Sterilization. — Sterilization  in  the  presence  of 
moisture  and  at  temperature  above  100°  C.,  requires  a 
pressure  greater  than  that  of  the  atmosphere  and  the  apparatus 
used  for  this  purpose  is  known  as  the  autoclave.  All  bacteria 
and  their  spores  are  killed  by  heating  at  110°  C.,  in  the  presence 
of  water,  for  fifteen  minutes,  and  in  about  five  minutes  at  120°  C. 
The  steam  pressures  corresponding  to  these  temperatures  are 
approximately  7 J  pounds  and  1 5  pounds  per  square  inch  or  J  kilo- 


STERILIZATION — ANTISEPSIS — FOOD   PRESERVATION 


69 


gram  and  i  kilogram  per  square  centimeter,  respectively.  The 
autoclave  consists  of  a  metal  cylinder  with  a  movable  top,  which  is 
fastened  down  tightly  during  sterilization.  It  is  furnished  with 
a  pressure  gauge,  a  stop-cock,  and  a  safety-valve  which  is  set 
to  allow  the  steam  to  escape  when  the  desired  pressure  is  attained 
and  thus  prevents  it  from  running  too  high.  Heat  is  furnished 
by  a  gas-burner  underneath .  The  lower 
part  of  the  cylinder  contains  water. 
The  objects  to  be  sterilized  are  sup- 
ported above  this  water  on  a  perforated 
bottom  or  shelf. 

%  It  is  necessary  to  follow  certain  pre- 
cautions in  the  use  of  the  autoclave. 
Allusion  has  already  been  made  to  the 
necessity  for  having  the  steam  saturated 
with  moisture.  This  is  effected  by 
allowing  the  air  to  escape  after  the  heat 
is  applied,  and  in  order  to  be  sure  that 
all  the  air  has  really  been  expelled,  the 
stop-cock,  with  which  all  autoclaves  are 
provided,  is  left  open  until  the  steam 
escapes  freely.  The  stop-cock  is  then 
closed,  and  the  pressure  begins  to  rise. 
After  leaving  the  articles  to  be  steril- 
ized in  the  autoclave  for  the  length  of 
time  desired,  the  apparatus  must  not  be 

opened  while  the  steam  contained  within  it  is  still  under  pressure, 
as  there  may  be  a  sudden  evolution  of  steam  upon  the  removal 
of  the  pressure  which  may  blow  the  media  out  of  their  tubes  and 
flasks.  After  the  pressure  has  fallen  to  zero  it  is  well  to  open  the 
stop-cock  only  a  little  way  so  that  air  may  not  be  drawn  in 
too  rapidly  to  replace  the  condensing  steam.  The  autoclave 
may  be  opened  as  soon  as  the  internal  and  external  pressure 
become  equal. 

The  length  of  exposure  necessary  to  accomplish  sterilization 


FIG.  32. — Autoclave. 


70  BACTERIOLOGY 

in  the  autoclave  depends  upon  the  protection  which  the  article 
to  be  sterilized  affords  the  bacteria.  In  sterilizing  agar,  a  con- 
siderable interval  elapses  before  the  agar  becomes  liquified,  es- 
pecially if  it  be  in  large  flasks,  and  it  is  well  to  allow  30  to  35 
minutes  at  110°  C.,  for  its  sterilizaton.  Closely  packed  surgical 
dressings  serve  to  protect  the  interior,  and  considerable  time  may 
be  required  for  penetration  of  a  sterilizing  temperature  into  such 
packages.  In  such  instances  it  is  unwise  to  rely  upon  the  gauge 
as  an  indicator  of  the  temperature  throughout  the  materials 
being  sterilized.  It  is  well  to  test  the  efficiency  of  the  steriliza- 
tion from  time  to  time  by  enclosing  test  objects  in  the  center  of 
several  packages.  A  convenient  test  object  for  surgical  auto- 
claves may  be  made  by  spreading  spores  of  B.  subtilis  or  B.  vulga- 
tus  on  a  sterile  cover-glass  and  placing  it  in  a  sterile  test-tube 
plugged  with  cotton,  and  then  drying  the  preparation  thor- 
oughly in  the  incubator  for  24  hours.  A  number  of  these  may  be 
prepared  and  subsequently  kept  in  the  refrigerator  until  used. 
After  the  test  object  has  been  exposed  in  the  autoclave,  sterile 
broth  is  added  to  the  tube  by  means  of  a  capillary  pipette.  The 
development  of  a  culture  from  the  spores  indicates  lack  of  effi- 
ciency in  the  process  of  sterilization. 

Discontinuous  or  fractional  sterilization  by  moist  heat  is  em- 
ployed to  sterilize  certain  kinds  of  culture  media,  more  especially 
blood  serum  and  gelatin,  which  are  likely  to  be  injured  by  heat- 
ing above  100°  C.,  or  by  prolonged  heating.  In  this  method  the 
medium  is  exposed  to  a  temperature  deemed  sufficient  to  kill  the 
vegetative  forms  of  bacteria  but  not  the  spores.  An  interval  is 
then  allowed  for  the  generation  of  these  spores,  whereupon  the 
heat  is  again  applied.  This  sequence  is  repeated  until,  according 
to  past  experience,  sterilization  may  be  regarded  as  almost  cer- 
tainly accomplished.  In  the  case  of  gelatin  steaming  (100°  C.) 
for  15  to  20  minutes  on  three  consecutive  days  is  the  usual 
practice;  with  inspissated  serum,  exposure  for  i  hour  at  60°  to 
70°  C.  on  six  successive  days  is  usually  sufficient.  These  methods 
are  applicable  only  to  media  in  which  spores  may  germinate  and 


STERILIZATION — ANTISEPSIS — FOOD      PRESERVATION  71 

they  may  fail  to  sterilize  even  in  case  of  such  materials,  es- 
pecially in  the  presence  of  rapidly  growing  spore-producing  bac- 
teria and  when  there  are  spores  of  anaerobic  bacteria  in  the 
material  to  be  sterilized.  On  this  account,  materials  sterilized 
in  this  way  should  not  be  injected  into  patients. 

Electricity  has  little  or  no  direct  demonstrable  germicidal  ac- 
tion. An  electric  current  may  generate  sufficient  heat  to  kill 
bacteria,  or  it  may  produce  powerful  germicides  by  electrolysis, 
such  for  example  as  acids  and  alkalies. 

Chemical  Agents, — Sterilization  by  means  of  chemicals  is 
not  employed  in  the  preparation  of  culture  media  because  of  the 
difficulty  of  removing  the  added  substance  after  the  desired  effect 
has  been  obtained.  It  is  necessary  in  every  case  to  consider 
the  other  effects  which  the  use  of  chemical  germicides  entails,  and 
their  usefulness  is  therefore  somewhat  more  limited  than  that  of 
the  physical  agents  for  sterilization.  Their  efficiency  is  also 
subject  to  great  variation  according  to  the  nature  of  the  materials 
with  which  they  come  in  contact.  Nevertheless  they  have  a 
very  important  place  in  practical  sterilization  and  disinfection. 

The  common  soaps,  and  more  particularly  green  soap,  have 
a  slight  germicidal  value,  and  this  in  conjunction  with  their  sol- 
vent action  upon  fats  and  protein,  and  the  mechanical  cleans- 
ing which  accompanies  their  use,  justifies  assigning  them  an 
important  place  among  the  chemical  disinfectants. 

Acids,  especially  those  which  are  strongly  dissociated,  are 
powerful  germicides.  Hydrochloric  acid  apparently  owes  its 
power  entirely  to  its  acidity,  and  in  fairly  weak  solution,  0.2  to 
i  .o  per  cent,  it  kills  vegetative  bacteria  in  a  short  time.  Strong 
sulphuric  acid  actually  carbonizes  organic  matter,  while  nitric 
acid  oxidizes  and  also  forms  special  combinations  with  protein, 
the  reactions  resulting  in  death  of  living  protoplasm.  Sulphurous 
acid  (sulphur  dioxide)  also  possesses  marked  germicidal  proper- 
ties, probably  due  to  oxidation  effects. 

Sulphur  dioxide  gas  has  been  employed  extensively  in  the 
fumigation  of  rooms,  and  is  usually  prepared  by  burning  sulphur. 


72  BACTERIOLOGY 

Much  difference  of  opinion  exists  regarding  the  value  of  it  as  a 
disinfectant.  The  spores  of  anthrax  are  not  killed  by  several 
days'  exposure  to  the  liquefied  gas.  Anthrax  and  other  bacilli 
are  destroyed  in  thirty  minutes  when  exposed  on  moist  threads 
in  an  atmosphere  containing  one  volume  per  centum  of  the  gas. 
An  exposure  of  twenty-four  hours  in  an  atmosphere  containing 
four  volumes  per  centum  of  the  gas  will  destroy  the  organisms 
of  typhoid  fever,  diphtheria,  cholera  and  tuberculosis.  The 
presence  of  moisture  greatly  enhances  the  activity  of  the  disin- 
fectant, owing  to  the  formation  of  the  more  energetic  sulphurous 
acid. 

For  the  destruction  of  insects,  such  as  mosquitoes,  this  agent 
is  superior  to  formaldehyde.  Its  application  for  this  purpose  is 
important  in  preventing  the  spread  of  yellow  fever  and  malaria. 

In  practice,  at  least  3  pounds  of  sulphur  per  1000  cubic  feet 
should  be  used,  and  moisture  must  be  present.  This  latter  re- 
quirement can  be  fulfilled  by  evaporating  several  quarts  of  water 
within  the  tightly  closed  room  just  prior  to  generating  the  gas. 
In  using  powdered  or  flowers  of  sulphur,  the  necessary  amount 
is  placed  on  a  bed  of  sand  or  ashes  in  an  iron  pot,  which  should 
rest  on  a  couple  of  bricks  in  a  pan  or  other  vessel  containing  an 
inch  or  two  of  water.  The  sulphur  is  ignited  by  means  of  some 
glowing  coals,  or  by  moistening  with  alcohol  and  applying  a 
match.  Difficulty  is  often  experienced  in  keeping  the  sulphur 
burning,  and  for  this  reason  it  is  surer  and  more  convenient  to 
use  the  so-called  sulphur  candles  now  on  the  market.  In  operating 
with  these,  a  sufficient  number  are  placed  on  bricks  in  a  pan  of 
water  and  the  wicks  lighted.  Liquefied  sulphur  dioxide  may  be 
used,  and  can  now  be  obtained  in  convenient  tin  receptacles  con- 
taining a  sufficient  quantity  for  the  disinfection  of  an  ordinary 
room.  The  can  is  opened  by  cutting  through  a  soft  metal  tube 
projecting  from  the  top.  The  fluid  vaporizes  at  the  room  tem- 
perature, and  it  is  simply  necessary  to  place  the  can  in  a  con- 
venient porcelain  dish  and  allow  the  fluid  to  evaporate. 

Sulphur  dioxide  is  objectionable  on  account  of  its  lack  of 


STERILIZATION — ANTISEPSIS — FOOD    PRESERVATION  73 

power  when  dry,  and  on  account  of  its  corrosive  action  on  metal 
and  its  bleaching  effect  on  hangings  and  draperies  in  the  presence^ 
of  moisture;  it  is,  therefore,  preferable  to  use  formaldehyde  for 
room  disinfection  when  possible. 

Alkalies,  especially  the  caustics,  sodium  hydroxide  and  potas- 
sium hydroxide,  are  powerful  germicides.  Commercial  lye  is  also 
valuable  as  a  disinfectant.  Perhaps  the  most  important  of  the 
alkalies  is  calcium  hydroxide,  Ca(OH)2  which,  because  of  its 
low  cost,  is  extensively  used  for  the  disinfection  of  excreta. 

Lime. — The  addition  of  o.i  per  cent  of  unslaked  lime  to  fluid 
cultures  of  the  typhoid  bacillus  and  cholera  spirillum  will  render 
them  sterile  in  four  or  five  hours.  Typhoid  dejecta  are  sterilized 
in  six  hours  when  thoroughly  mixed  with  3  per  cent  of  slaked  lime; 
the  addition  of  6  per  cent  will  accomplish  the  same  result  in  two 
hours.  A  convenient  form  for  practical  use  is  an  aqueous  mix- 
ture containing  20  per  cent  of  lime — so-called  milk  of  lime. 
Typhoid  and  cholera  dejecta  are  sterilized  in  one  hour  after  mix- 
ing with  20  per  cent  of  this  mixture.  In  practice  it  is  safer  to  use 
a  considerable  excess  of  lime.  From  the  foregoing  facts  it  would 
seem  probable  that  lime  or  whitewash  as  ordinarily  applied  would 
possess  disinfectant  properties.  Experimental  work  has  demon- 
strated this  to  be  a  fact.  The  organisms  of  anthrax,  glanders  and 
the  pus  cocci  were  destroyed  within  twenty-four  hours  by  one 
application.  For  spore-forming  organisms  and  the  bacillus  of 
tuberculosis  the  power  is  not  so  great,  the  latter  organism  not 
being  destroyed  by  three  applications  of  the  whitewash.  Practi- 
cally, whitewashing  is  an  effective  means  of  disinfecting  wood- 
work, perhaps  because  those  microbes  which  are  not  killed  at  once 
are  caught  in  the  whitewash  and  their  further  distribution 
prevented. 

Oxidizing  agents  are  usually  germicidal.  Chlorine,  bromine 
and  iodine,  ozone,  nitric  acid,  potassium  permanganate,  chlorinated 
lime,  organic  peroxides  and  peracids,  and  hydrogen  peroxide, 
belong  to  this  class.  Chlorine,  employed  as  chlorinated  lime, 
is  a  valuable  disinfectant  for  excreta.  In  the  form  of  bleaching 


74  BACTERIOLOGY 

powder  it  has  been  extensively  used  in  the  disinfection  of  drinking 
water  and  of  swimming  pools.  Bromine  and  iodine  have  long 
been  employed  in  surgery,  and  solutions  of  iodine  are  often  applied 
to  the  skin  before  surgical  incision.  Iodine  probably  acts  to 
some  extent  as  a  germicide  in  this  instance,  but  also  as  an  anti- 
septic, remaining  in  the  skin  for  some  time  after  its  application. 
Hydrogen  peroxide  is  a  germicide,  as  it  quickly  decomposes  to 
form  water  and  oxygen.  It  is  placed  on  the  market  in  solutions 
varying  in  strength  from  10  to  30  volumes,  the  mode  of  expression 
indicating  that  corresponding  solutions  will  liberate  ten  to  thirty 
times  their  volume  of  oxygen  when  appropriately  treated.  It 
decomposes  rapidly  when  in  contact  with  purulent  secretions, 
setting  free  abundant  oxygen,  and  on  this  account  is  much  used 
for  cleansing  infected  wounds.  It  deteriorates  in  strength  so 
rapidly  that  only  fresh  solutions  of  known  strength  should  be 
used. 

Potassium  Permanganate. — Koch  asserts  that  a  3  per  cent 
solution  will  destroy  anthrax  spores  in  twenty-four  hours,  but 
that  a  i  per  cent  solution  cannot  be  depended  upon  to  kill  patho- 
genic organisms.  Its  disinfectant  value  in  practice  is  very  low 
on  account  of  its  ready  decomposition  by  inert  material.  In  the 
dilute  solutions  usually  used  for  medicinal  injections  and  irriga- 
tions no  disinfectant  action  occurs. 

lodoform. — This  substance  possesses  little  if  any  disinfectant 
power.  It  is  mildly  antiseptic  in  moist  wounds,  due  to  the  gradual 
liberation  of  small  quantities  of  iodine. 

Inorganic  Salts. — Mercuric  chloride,  HgC^,  is  probably  more 
commonly  used  than  any  other  one  germicide.  But  Geppert, 
whose  work  in  this  direction  has  been  abundantly  corroborated 
by  others,  found  that  the  potency  of  corrosive  sublimate  as  a 
germicide  had  been  greatly  overrated.  The  inhibitory  action  of 
corrosive  sublimate,  on  the  other  hand,  is  very  great,  and  the 
veriest  trace  of  it  left  adhering  to  the  bacteria  is  sufficient  to 
prevent  them  from  growing.  Corrosive  sublimate  is  difficult 
to  remove  by  ordinary  washing  and  traces  of  it  remain  even  after 


STERILIZATION — ANTISEPSIS — FOOD    PRESERVATION  75 

very  thorough  washing.  But  if  the  last  traces  are  removed  by 
treatment  with  ammonium  sulphide  or  other  reagents  which  pre^ 
cipitate  the  mercury  salt  without  themselves  injuring  the  bac- 
teria, growth  takes  place  even  where  the  corrosive  sublimate 
solutions  have  been  used  which  are  apparently  efficacious.  Thus 
anthrax  spores  will  not  grow  in  culture  media  when  they  are 
exposed  for  even  a  few  minutes  on  silk  threads  to  the  action  of 
corrosive  sublimate  solution  of  the  strength  of  yV  per  cent  and 
then  washed  thoroughly  in  water  and  rinsed  in  alcohol;  but 
Geppert  showed  that  the  spores  so  treated  were  only  apparently 
killed,  for  it  took  twenty  hours'  exposure  to  corrosive  sublimate 
solution  of  this  strength  where  the  spores  were  not  dried  on  silk 
threads,  but  suspended  in  water,  and  where  the  last  trace  of 
corrosive  sublimate  was  removed  by  treatment  with  ammonium 
sulphide.  It  is  claimed  that  its  affinity  for  albuminous  bodies 
and  the  readiness  with  which  it  combines  with  such  substances 
detract  from  its  value  for  some  purposes.  On  the  other  hand, 
many  observers  claim  that  the  albuminous  combinations  formed 
under  such  circumstances  are  soluble  in  an  excess  of  albuminous 
fluid,  and  that  its  value  as  a  germicide  is  not  affected  thereby. 
To  obviate  this  possible  difficulty  it  is  customary  in  practice  to 
combine  the  bichloride  of  mercury  with  some  substance  that  will 
prevent  the  precipitation  of  the  mercury  salt  by  albumin.  For 
this  purpose  5  parts  of  any  one  of  the  following  substances  to  i 
part  of  bichloride  of  mercury  may  be  used — hydrochloric  acid, 
tartaric  acid,  sodium  chloride,  potassium  chloride,  or  ammonium 
chloride.  A  very  practical  stock  solution  for  laboratory  purposes 
has  the  following  composition: 

Hydrochloric  acid 100  c.c. 

Bichloride  of  mercury 20  grams. 

Five  c.c.  in  a  liter  of  water  makes  a  solution  of  about  i-iooo  strength. 

Mercuric  Iodide. — An  extremely  high   antiseptic  value  has 
been  placed  on  this  substance  by  Miquel,  who  claims  that  the 


76  BACTERIOLOGY 

most  resistant  spores  are  prevented  from  developing  in  a  cul- 
ture medium  containing  1-40,000.  In  combination,  as  potas- 
sio-mercuric  iodide,  it  has  been  used  in  soaps  (McClintock) 
with  very  favorable  results.  The  substance  is  not  extensively 
employed,  and  further  investigation  is  necessary  to  determine 
its  true  value. 

Silver  Nitrate. — This  salt  probably  occupies  the  next  posi- 
tion to  the  bichloride  of  mercury  in  germicidal  power.  Behr- 
ing  claims  it  to  be  superior  to  bichloride  of  mercury  in  albumin- 
ous fluids.  The  anthrax  bacillus  is  killed  by  a  solution  of 
1-20,000  after  two  hours'  exposure.  At  least  forty-eight  hours' 
exposure  to  a  1-10,000  solution  is  required  to  kill  the  spores  of 
anthrax.  It  is  very  irritating,  and  possesses  strong  affinities 
for  chlorides,  forming  with  them  insoluble  chloride  of  silver,  a 
salt  without  germicidal  value.  For  these  reasons  the  use  of 
silver  nitrate  is  limited.  In  the  solutions  usually  employed  for 
douching  the  cavities  of  the  body  the  available  silver  nitrate  is 
immediately  converted  into  the  insoluble  chloride,  and  little  if 
any  germicidal  action  takes  place.  To  this  fact  may  be  ascribed 
the  varying  clinical  results  reported. 

Many  proprietary  silver  compounds  are  on  the  market,  in- 
troduced to  replace  the  nitrate  and  its  objectionable  features. 
The  most  important  are  protargol  and  argyrol,  organic  silver 
combinations.  They  do  not  combine  with  chlorides,  are  less 
irritating  than  the  nitrate  and,  not  coagulating  albumin,  they 
possess  greater  penetrating  power. 

Organic  Poisons. — Carbolic  acid  is  one  of  the  most  important 
and  most  widely  used  disinfectants.  It  is  usually  employed  in 
strengths  of  from  i  to  5  per  cent.  A  3  per  cent  solution  will 
sometimes  kill  the  spores  of  anthrax  after  two  days'  exposure. 
In  the  absence  of  spores,  the  anthrax  bacillus  is  destroyed  by  a  i  per 
cent  solution  in  one  hour.  The  less  resistant  pus  cocci  are  de- 
stroyed rapidly  by  a  2  per  cent  solution.  Combination  with  an 
equal  proportion  of  hydrochloric  acid  enhances  the  efficacy  of 
carbolic  acid  to  a  marked  extent.  This_is  due  to  the  prevention 


STERILIZATION — ANTISEPSIS FOOD    PRESERVATION  77 

of  albuminous  combinations,  thus  allowing  greater  penetration 
of  the  disinfectant. 

Many  other  substances  closely  related  to  carbolic  acid  are 
used  and  possess  marked  germicidal  properties.  Among  them 
may  be  mentioned  creolin,  cresol  and  lysol.  They  are  all  slightly 
superior  to  carbolic  acid  in  actual  germicidal  value. 

Formalin  is  a  40  per  cent  aqueous  solution  of  formaldehyde, 
H2CO.  Remarkable  claims  have  been  made  for  this  substance, 
and  numerous  investigations  have  shown  it  to  possess,  both  in 
the  liquid  and  gaseous  forms,  wonderful  disinfecting  power  under 
certain  conditions.  Later  investigations  indicate  that  its  germi- 
cidal power  had  been  somewhat  overestimated.  In  solutions 
of  i-iooo  an  exposure  of  twenty-four  hours  is  necessary  to  destroy 
the  staphylococcus  pyogenes  aureus,  while  1-5000  is  sufficient 
to  restrain  its  growth  (Slater  and  Rideal).  Its  use  in  a  gaseous 
form  as  a  house  disinfectant  is  by  far  the  most  important  applica- 
tion at  the  present  time. 

From  250  to  500  c.c.  of  formalin  together  with  500  to  1000  c.c. 
of  water  should  be  vaporized  for  each  1000  cubic  feet  of  air  space 
in  the  room,  and  the  room  should  remain  tightly  closed  for  at 
least  four  hours,  preferably  over  night.  Many  methods  of 
vaporizing  formaldehyde  have  been  devised.  Some  form  of 
tank,  provided  with  heating  apparatus  and  with  an  outlet  tube 
which  passes  through  the  keyhole  into  the  room,  is  perhaps  the 
most  convenient  where  much  disinfection  has  to  be  done.  If 
apparatus  of  this  sort  is  not  at  hand,  good  results  may  be  obtained 
by  putting  the  formalin  and  the  water  previously  heated  to  boil- 
ing, in  a  large  pail  in  the  center  of  the  room,  and  then  adding 
rapidly  crystalline  potassium  permanganate,  about  200  grams  to 
each  500  c.c.  of  formalin  used.  The  permanganate  oxidizes 
some  of  the  formaldehyde  and  produces  heat  to  evaporate  the 
rest  of  it.  From  25  to  50  per  cent  more  formalin  should  therefore 
be  used  for  a  given  air  space.  It  is  well  also  to  add  about  10  per 
cent  of  glycerin  to  the  water  so  as  to  raise  the  boiling-point  some- 
what and  insure  more  complete  vaporization  of  the  formaldehyde. 


78  BACTERIOLOGY 

Formaldehyde  penetrates  very  slightly  beneath  exposed 
surfaces  so  that  everything  to  be  disinfected  should  be  completely 
exposed.  Openings  about  windows  and  doors  should  be  carefully 
plugged  up  and  sealed  with  strips  of  paper.  Mechanical  cleansing 
supplemented  by  application  of  i-iooo  solution  of  mercuric 
chloride  to  floors  and  walls  should  follow  the  fumigation.  The 
persistent  odor  of  formalin  may  be  removed  by  fumes  of 
ammonia. 

Aniline  Dyes. — Many  of  the  aniline  dyes,  notably  pyoktanin 
(methyl- violet) ,  possess  germicidal  properties.  Malachite  green 
is  said  to  possess  even  greater  germicidal  value  than  pyoktanin. 
Methylene  blue  also  possesses  considerable  germicidal  power. 

Alcohol  is  a  germicide  of  moderate  power.  It  has  little 
effect  upon  spores  but  in  concentrations  of  from  50  to  95  per  cent 
it  destroys  vegetative  bacteria  in  a  few  minutes. 

Germicides  destroy  bacteria,  as  a  general  rule,  because  they 
are  general  protoplasmic  poisons,  destructive  to  all  living  matter. 
There  is,  nevertheless,  some  selective  action.  Thus,  formal- 
dehyde kills  bacteria  but  has  little  poisonous  effect  upon  insects, 
such  as  mosquitoes,  bedbugs,  roaches  or  fleas.  Mercuric  chloride 
is  rapidly  fatal  to  bacteria  when  it  comes  into  contact  with  them, 
but  it  has  no  very  immediate  destructive  effect  upon  fly  larvae 
(maggots).  Some  of  the  oxidizing  agents,  such  as  hydrogen 
peroxide  and  acetozone  are  not  poisonous  to  man  because  they 
are  decomposed  into  relatively  harmless  substances  before  they 
can  be  absorbed.  Attempts  to  discover  or  to  produce  chemicals 
which  would  exhibit  a  selective  destructive  effect  upon  microbes 
in  the  interior  of  the  body  have  not  met  with  much  success. 
Quinine  is  perhaps  the  best  known  example,  as  it  may  circulate 
in  the  blood  in  sufficient  concentration  to  poison  the  malarial 
parasites  without  at  the  same  time  killing  the  host.  The  effects 
produced  by  mercury  and  by  salvarsan  in  syphilis  are  perhaps 
analogous,  but  they  evidently  depend  to  a  large  extent  upon  a 
special  susceptibility  of  the  microbe,  a  susceptibility  not  yet 
apparent  in  most  parasites.  The  specific  immune  substances 


STERILIZATION — ANTISEPSIS — FOOD    PRESERVATION  79 

may  perhaps  be  classed  in  the  same  category.  These  will  be 
considered  in  more  detail  in  a  later  chapter. 

Antiseptics. — Antiseptic  and  preservative  agents  prevent  or 
delay  the  development  of  bacteria,  without  killing  them.  Very 
much  the  same  agents  are  applied  to  prevent  the  growth  of  mi- 
crobes in  living  tissues  and  consequent  poisoning  of  the  body 
(antisepsis)  as  in  preventing  microbic  development  in  dead 
organic  matter  (food  preservation). 

Of  the  physical  antiseptics,  dessication  and  cold  are  perhaps 
of  greatest  importance.  These  agencies  find  application  to  the 
living  body  as  well  as  in  preservation  of  dead  material.  Sub- 
stances which  increase  osmotic  pressure,  sodium  chloride  and 
sugar,  are  also  employed  to  prevent  microbic  growth  in  foods. 

The  chemical  antiseptics  are  very  numerous.  In  general 
a  germicide  in  higher  dilution  exhibits  antiseptic  effect.  *  Small 
quantities  of  the  inorganic  acids,  hydrochloric,  nitric,  sulphuric 
or  sulphurous  acid,  prevent  bacterial  growth.  Even  boric  acid 
which  has  little  or  no  germicidal  effect  will  delay  or  inhibit  mi- 
crobic development.  Many  organic  acids  possess  inhibitive  prop- 
erties toward  bacterial  action.  Acetic  and  lactic  acids  prob- 
ably act  merely  by  virtue  of  their  acidity.  Benzoic  and  salicylic 
acids  seem  to  be  more  antiseptic,  probably  by  virtue  of  other 
structural  features  in  their  molecules.  Other  organic  substances, 
such  as  phenol  (carbolic  acid)  and  formaldehyde  in  high  dilu- 
tions prevent  or  delay  bacterial  growth,  and  weaker  germicides 
such  as  alcohol,  chloroform  or  ether,  are  fairly  effective  preserva- 
tives. Oxidizing  agents  often  decompose  too  rapidly  to  be  of 
much  value  as  antiseptics.  Iodine,  however,  is  one  member  of 
this  group  having  considerable  antiseptic  value. 

Of  the  inorganic  salts,  mercuric  chloride  is  most  important. 
Small  quantities  of  this  agent  inhibit  the  multiplication  of  bac- 
teria. It  is  extensively  employed  in  antiseptic  treatment  of 
wounds.  The  borates,  nitrates  and  salicylates,  the  latter  com- 
pounds of  an  organic  acid,  also  inhibit  bacterial  action  to  some 
extent. 


8o  BACTERIOLOGY 

In  using  these  substances  as  antiseptic  applications  to  wounds, 
the  possible  poisonous  effects  upon  the  body  as  a  whole  from 
absorption  of  the  antiseptic  must  be  kept  in  mind.  Moreover, 
such  substances  ought  not  to  be  used  as  food  preservatives  with- 
out due  regard  to  the  changes  they  may  induce  in  the  food  and 
the  possible  effects  they  may  exert  upon  the  consumer. 

TESTING  ANTISEPTICS  AND  DISINFECTANTS. 

The  determination  of  the  antiseptic  value  of  a  material  is  a 
comparatively  simple  matter.  A  virulent  culture  of  the  organ- 
ism used  as  a  test  is  inoculated  into  sterile  bouillon  containing  a 
known  quantity  of  the  antiseptic.  The  process  is  repeated  with 
varying  strengths  of  the  material  until  the  smallest  quantity  of 
it  capable  of  preventing  growth  is  determined.  This  dilution 
may  be  considered  the  antiseptic  value  of  the  material  in  question 
for  the  organism  used,  under  the  conditions  of  the  test. 

Determination  of  the  disinfectant  power  of  a  substance  is  a 
problem  of  much  greater  magnitude,  and  the  method  used  must 
be  altered  more  or  less  to  suit  the  properties  of  the  substance 
tested.  It  is  obvious  that  some  of  the  substance  tested  remains 
in  contact  with  the  organisms  in  the  method  given  for  determin- 
ing the  antiseptic  value,  and  that  we  do  not  know  whether  the 
bacteria  are  alive  and  merely  inhibited  in  growth,  or  actually 
killed. 

The  chemical  composition  of  the  medium  in  which  the  bac- 
teria are  tested  may  have  a  marked  influence  upon  the  action  of 
germicides.  If  components  of  the  medium  enter  into  chemical 
union  with  the  germicide  there  may  be  an  inert  compound 
formed.  There  may  also  be  formed  dense,  flocculent  precipi- 
tates which  envelop  the  bacteria  and  protect  them  from  the  action 
of  the  germicide.  It  is  therefore  apparent  that  the  potency  of  a 
germicide  may  appear  very  different  when  acting  upon  the  bac- 
teria in  water  or  in  physiological  salt  solution  or  on  bacteria 
dried  on  glass  rods  or  on  silk  threads,  on  the  one  hand,  and  upon 


STERILIZATION — ANTISEPSIS — FOOD    PRESERVATION  8 1 

the  same  bacteria  in  beef  broth  or  in  feces  or  in  urine,  on  the  other. 
For  these  reasons  it  is  not  always  possible  to  draw  conclusions 
from  the  results  of  laboratory  experiments  as  to  the  value  of  a 
germicidal  agent  for  practical  disinfecting  purposes. 

Method. — To  15  c.c.  of  sterile  water  in  a  60  c.c.  Erlenmeyer 
flask  add  2  c.c.  of  a  virulent  culture  of  the  test-organism.  Then 
add  a  solution  of  the  substance  under  investigation  in  the  pro- 
portion necessary  to  give  the  dilution  wished.  Mix  thoroughly, 
and  allow  this  "  action-flask "  to  stand  as  long  as  it  is  desired  to 
have  the  germicide  in  contact  with  the  test-organism  (action- 
period).  Transfer  0.5  c.c.  from  the  action-flask  to  a  flask  con- 
taining 200  c.c.  of  a  solution  of  some  chemical  capable  of  decom- 
posing the  substance  being  tested  with  the  formation  of  inert  or 
insoluble  compounds.  In  this  "  inhibition-flask "  the  strength 
of  the  solution  should  be  such  that  molecular  proportions  of  the 
chemical  are  present  in  sufficient  quantity  to  combine  with  all 
the  germicide  carried  over.  The  inhibition-flask  is  shaken  for 
30  seconds,  and  i  c.c.  transferred  from  it  to  100  c.c.  of  sterile 
water  in  another,  the  "  dilution-flask."  After  two  minutes, 
three  agar  tubes  are  inoculated  with  i  c.c.  each  from  the  dilution- 
flask,  plated,  and  growth  watched  for. 

Control-experiments  should  be  performed  to  determine  that 
the  dilution  of  the  test-culture  is  not  too  great  when  carried  through 
the  three  flasks.  It  likewise  should  be  determined  that  the  in- 
hibiting chemical  has  no  effect  on  the  bacteria. 

What  the  inhibiting  chemical  shall  be  must  be  determined 
for  each  individual  case.  For  salts  of  the  heavy  metals  ammo- 
nium sulphide  answers  well;  for  mercury  salts,  stannous  chloride 
may  be  used;  for  formaldehyde,  ammonium  hydrate;  for  carbolic 
acid,  sodium  sulphate. 

The  testing  of  gaseous  disinfectants,  such  as  sulphur  dioxide 
and  formaldehyde,  must  be  conducted  under  conditions  as  nearly 
parallel  to  actual  practice  as  possible.  The  test-organisms  may 
be  exposed  on  threads  or  cover-glasses,  and  acted  upon  by  a  known 
volume  strength  of  disinfectant 'for  a  known  length  of  time. 
6 


82  BACTERIOLOGY 

Subsequent  treatment  of  the  organisms  with  a  suitable  inhibitor 
is  necessary  when  possible,  and  should  growth  occur  in  the  cul- 
tures following,  the  test-organism  should  be  recognized  in  order 
that  possible  contamination  by  extraneous  organisms  may  be 
excluded. 

In  determining  the  value  of  germicides  for  sterilizing  ligatures, 
the  students  can  apply  methods  based  on  the  foregoing  principles. 
Great  care  and  ingenuity  are  necessary  to  arrive  at  correct  con- 
clusions, particularly  in  the  case  of  animal  tendons.  In  many 
instances  quite  stable  compounds  are  formed  between  tendon 
and  germicide,  and  living  organisms  may  be  so  imbedded  in  such 
a  substance  that  subsequent  growth  in  a  test-culture  is  impossible. 
The  use  of  a  suitable  inhibitor,  and,  prior  to  final  culture-tests, 
a  prolonged  soaking  in  sterile  water,  will  promote  the  accuracy 
of  the  results. 


CHAPTER  III. 
CULTURE  MEDIA. 

Culture  media  are  substances  in  which  microbes  are  artificially 
cultivated.  The  variety  of  such  substances  is  very  large,  different 
materials  being  suited  to  different  purposes.  Particular  kinds 
of  media  have  been  devised  in  order  to  bring  to  development  or 
especially  to  favor  the  development  of  certain  kinds  of  microbes. 
Various  media  are  also  used  to  demonstrate  the  physiological 
properties  of  bacteria,  especially  the  physical  arrangement  of  the 
bacterial  cells  as  they  grow  under  various  conditions,  and  the 
chemical  changes  induced  in  the  various  constituents  of  the 
media  by  the  microbic  growth. 

Glassware. — Micro-organisms  are  usually  grown  in  glass 
test-tubes  or  sometimes  in  glass  flasks.  The  tubes  and  flasks 
should  be  of  more  durable  glass  than  those  ordinarily  used  in 
chemical  work,  but  heavy  tubes  of  glass  of  poor  quality  are  not 
to  be  recommended.  For  ordinary  purposes,  test-tubes  I25X 
15  mm.  are  convenient.  Larger  tubes,  150X20  mm.,  are  used 
to  store  media  to  be  used  in  making  plate  cultures  and  for  roll- 
tube  cultures.  New  glassware  should  be  thoroughly  washed 
before  using,  and  for  critical  work  it  should  be  boiled  in  dilute 
sodium  carbonate,  rinsed,  washed  in  dilute  hydrochloric  acid, 
rinsed  repeatedly  in  running  water,  finally  in  distilled  water, 
and  then  inverted  to  drain  in  a  warm  place,  such  as  the  incubator, 
until  perfectly  dry.  Used  glassware  should  be  sterilized  in  the 
autoclave  at  120°  C.  for  half  an  hour,  emptied,  cleaned  with  a 
swab  and  hot  water,  rinsed  in  distilled  water  and  drained.  In 
case  of  special  difficulty  the  glassware  may,  after  emptying  and 
washing  in  water,  be  cleaned  by  soaking  in  a  special  cleaning  fluid, 
and  all  organic  matter  may  be  readily  removed  by  using  this 

83 


84  BACTERIOLOGY 

fluid  hot.  It  should  not  come  into  contact  with  the  hands  or 
with  any  large  quantity  of  organic  matter. 

CLEANING  FLUID. 

Potassium  or  sodium  bichromate 40  grams. 

Water 150  c.c. 

Dissolve  the  bichromate  in  water,  with  heat; 
allow  it  to  cool;  then  add,  carefully,  con- 
centrated commercial  sulphuric  acid 230  c.c. 

Exact  proportions  are  not  necessary  in  making  this  fluid.  Glass- 
ware cleaned  in  it  must  be  repeatedly  rinsed  subsequently. 

Plugs. — The  clean  dry  tubes  or  flasks  are  plugged  with  raw 
cotton  of  a  good  grade  which  does  not  char  too  readily  upon 
heating.  The  cotton  plugs  may  be  carefully  made  by  rolling 
an  oblong  rectangular  strip,  of  even  thickness,  into  a  firm  cylinder 
of  proper  size,  rolled  plugs,  or  more  hastily  made  by  stuffing  the 
cotton  into  the  open  end  of  the  flask  or  tube,  stuffed  plugs.  The 
latter  kind  of  plug  serves  very  well  for  tubes  in  which  media  are 
to  be  stored  temporarily  but  is  not  so  satisfactory  for  other 
purposes. 

Sterilization. — After  plugging,  the  tubes  are  placed  in  a  wire 
basket  and  sterilized  in  the  hot-air  sterilizer  or,  sometimes,  to 
avoid  charring,  in  the  autoclave.  This  not  only  renders  the 
glassware  free  from  bacteria  but  also  gives  more  permanent 
form  to  the  plugs. 

THE  COMMON  CULTURE  MEDIA. 

Broth. — Broth,  bouillon  or  beef- tea,  is  best  made  from  fresh 
meat,  either  beef,  veal  or  chicken.  Finely  chopped  lean  meat,  450 
to  500  grams,  is  mixed  with  1000  c.c.  of  distilled  water  and  either 
allowed  to  stand  over  night  in  the  refrigerator  or  else  digested 
for  half  an  hour  at  temperature  of  50  to  55°  C.  It  is  then  strained 
through  muslin,  yielding  a  filtrate  of  deep  red  color.  Any  ex- 
cessive amount  of  fat  should  be  skimmed  off.  To  the  filtrate, 
which  should  measure  1000  c.c.,  are  added: 


CULTURE    MEDIA  85 

Peptone,  Witte's1 10  grams. 

Sodium  chloride  (common  salt) 5  grams. 

These  should  be  dissolved  by  stirring  at  a  temperature  below 
60°  C.  The  mixture  is  then  boiled  for  half  an  hour  over  the 
direct  flame,  cooled  slightly,  and  filtered  through  paper  pre- 
viously wet  with  warm  water.  The  filtrate  should  be  clear  and 
light  yellow  in  color,  and  should  be  diluted  to  1000  c.c.  with 
distilled  water.  Its  reaction  is  acid,  a  reaction  unfavorable  to 
the  growth  of  many  bacteria,  especially  to  many  pathogenic, 
forms. 

The  amount  of  alkali  to  be  added  is  ascertained  by  titration. 
For  this  purpose  exactly  5  c.c.  of  the  broth  is  placed  in  each  of 
three  test-tubes.  Five-tenths  cubic  centimeters  of  a  5  per  cent 
solution  of  purified  litmus  (Merck's  highest  purity)  is  added  to 

N 

each  tube.  An  accurately  prepared  —  solution  of  sodium  hy- 
droxide2 is  then  run  in  drop  by  drop  from  a  graduated  burette, 
the  reading  of  which  has  been  recorded,  into  one  of  the  tubes 
until  the  red  color  just  changes  to  blue.  The  burette  reading  is 
taken  and  recorded.  The  alkali  is  then  run  into  the  second  tube 
rather  rapidly  until  the  endpoint  ascertained  by  the  first  test  is 
nearly  reached.  By  comparing  the  color  of  this  tube  with  that  of 
the  first  one  and  with  the  third  to  which  no  alkali  has  yet  been 
added,  the  exact  point  at  which  the  color  is  changing  from  red 
to  blue  may  be  accurately  judged.  When  this  point  is  reached, 
the  burette  reading  is  again  recorded  and  the  amount  of  alkali 
necessary  to  neutralize  the  5  c.c.  of  broth  ascertained.  The 
third  tube  should  then  be  titrated  to  confirm  the  previous  result. 
The  titration  of  the  broth  should  now  be  repeated,  using  phe- 
nolphthalein  as  an  indicator.  For  this  purpose,  5  c.c.  of  the  medium 
is  transferred  to  a  small  porcelain  dish,  diluted  by  the  addition 

1  Commercial  peptones  are  mixtures  of  albumoses  and  a  small  amount  of  peptone. 

2  A  normal  solution  of  sodium  hydroxide  contains  one  gram-molecule  of  anhy- 
drous NaOH,  or  40  grams,  in  a  liter.     A  —  solution    contains  -%$  of  this  amount 
or  2  grams  in  a  liter. 


86  BACTERIOLOGY 

of  approximately  45  c,c.  of  distilled  water,  and  boiled  for  a  minute, 
i  c.c.  of  a  0.5  per  cent  solution  of  phenolphthalein  in  50  per  cent 

N 

alcohol  is  now  added  and  —  solution  of  sodium  hydroxide  run  in 

20 

from  the  burette  until  the  color  changes  to  a  faint  but  distinct 
and  permanent  pink  color.  The  burette  reading  is  recorded 
and  the  amount  of  alkali  necessary  to  neutralize  the  5  c.c.  of 
medium  in  respect  to  phenolphthalein  thus  ascertained.  This 
titration  may  well  be  repeated,  especially  by  beginners.  As  a 
result  of  these  titrations  we  shall  have  ascertained  the  amount  of 
alkali  necessary  to  neutralize  the  remaining  broth  to  either  indi- 
cator. For  example  suppose  that  5  c.c.  of  the  broth  titrated  as 
follows : 

0.5  c.c.  of  — alkali  with  litmus  as  indicator. 

20 

2.  o  c.c.  of  — alkali  with  phenolphthalein  as  indicator 

In    order    to    neutralize    the    remaining    980    c.c.   of   broth   to 

litmus  would  require  —     —  or  08  c.c.  of         alkali.      A  solu- 

5  20 

tion  of  alkali  twenty  times  as  strong  as  this,  namely  normal 
sodium  hydroxide,  is  employed  for  this  purpose,  and  only  f £  or 
4.9  c.c.  of  this  are  necessary  to  neutralize  the  980  c.c.  of  broth 
to  litmus.  The  reaction  generally  required  for  pathogenic 
bacteria  is  slightly  alkaline  to  litmus  and  for  this  reason  an  excess 
of  10  c.c.  of  normal  alkali  per  liter  is  added  to  the  broth,  9.8  c.c. 
for  the  980  c.c.,  making  altogether  14.7  c.c.  to  be  added.  Cal- 
culation from  the  result  obtained  with  phenolphthalein  in  the 
same  way  shows  that  19.6  c.c.  of  normal  alkali  would  be  required 
to  neutralize  the  medium  to  this  indicator.  The  desired  final 
reaction  of  the  medium  in  respect  to  phenolphthalein  is  acid, 
usually  that  of  5  to  15  c.c.  of  normal  acid  per  liter,  or  0.5  to  1.5 
per  100  c.c.,  or  0.5  to  1.5  per  cent,  as  it  is  commonly  expressed 
after  Fuller.1  In  this  instance,  therefore,  5  to  15  c.c.  per  liter, 
or  4.9  to  14.7  c.c.  less  than  the  19.6  for  the  980  c.c.,  would  be 

1  Fuller.     Journal  of  Amer.  Public  Health  Assoc.,  1905. 


CULTURE   MEDIA  87 

added,  namely  14.7  to  4.9  c.c.,  according  to  the  purpose  for  which 
the  broth  is  to  be  used. 

The  amount  of  normal  alkali  finally  decided  upon  is  added  to 
the  broth,  which  is  then  weighed  in  its  pan.  It  is  then  cooked 
by  boiling  over  the  direct  flame  for  half  an  hour  or  by  heating 
in  the  autoclave  at  110°  C.  for  15  to  20  minutes.  It  is  now 
cooled  to  about  50°  C.,  filtered  through  paper,  filled  into  tubes 
and  sterilized,  either  in  the  autoclave  at  110°  C.  for  15  minutes 
or  by  fractional  sterilization  in  streaming  steam  at  100°  C.  for 
15  minutes  on  three  consecutive  days. 

Broth  may  be  prepared  from  meat  extract  instead  of  meat. 
Meat  extract  3  grams,  peptone  10  grams  and  salt  5  grams  are 
dissolved  in  1000  c.c.  of  water,  boiled,  filtered  and  titrated  against 

N 

—  sodium  hydroxide.     The  subsequent  steps  are  the  same  as  in 

preparation  of  broth  from  fresh  meat. 

Remarks  upon  Titration. — The  titration  of  bacteriological 
media  made  from  meat  or  meat  extract  is  an  important  step  in 
their  preparation.  There  is  some  confusion  on  this  point  because 
of  the  use  of  different  indicators  in  ascertaining  the  reaction. 
The  neutral  point  indicated1  by  litmus  is  very  nearly  the  actual 
neutral  point  in  respect  to  acidity  and  alkalinity,  and  this  point 
is  not  appreciably  displaced  in  either  direction  by  the  addition 
of  a  neutral  mixture  of  a  feebly  dissociated  acid  and  its  salts  to 
the  solution.  The  end  reaction  indicated  by  phenolphthalein 
when  it  turns  pink  is  actually  a  point  at  which  there  is  a  slight 
excess  of  alkali.  This  is  so  nearly  the  actual  neutral  point  in 
inorganic  solutions,  when  electrolytic  dissociation  is  marked, 
that  the  error  is  not  appreciable.  In  solutions  of  organic  sub- 
stances, especially  when  considerable  amounts  of  feebly  dissoci- 
ated substances,  such  as  are  contained  in  peptone  or  gelatin,  are 
present,  this  error  becomes  very  appreciable.  The  discrepancy 
between  the  end  point  for  litmus  and  for  phenolphthalein  will 

1  Washburn,  E.  W.,  The  significance  of  the  term  alkalinity  in  water  analysis  and 
the  determination  of  alkalinity  by  means  of  indicators.  Report  Illinois  Waterworks 
Association,  191 1. 


88  BACTERIOLOGY 

vary  for  different  lots  of  media.  Another  source  of  error  and 
misunderstanding  arises  from  the  fact  that  the  reaction  of  a 
medium  changes  somewhat  after  its  neutralization,  especially 
during  sterilization,  but  also  upon  standing  afterward  at  ordinary 
temperature.  This  change  is  toward  decreased  alkalinity  and 
increased  acidity  and  its  extent  is  not  the  same  for  different 
media,  being  most  marked,  perhaps,  in  those  rich  in  glucose. 
Where  particular  importance  is  attached  to  the  titre  of  a  medium, 
it  is  well,  therefore,  to  determine  this  upon  a  sample  of  the  medium 
taken  from  the  lot  at  the  time  it  is  used,  rather  than  to  quote 
figures  obtained  before  sterilization.  The  optimum  reaction  for 
most  microbes  is  very  close  to  the  neutral  point  for  litmus  and 
preferably  slightly  alkaline  to  this  indicator. 

Gelatin. — Finely  chopped  meat,  450  to  500  grams,  is  mixed 
with  a  liter  of  distilled  water  and  digested  on  the  water  bath  for 
half  an  hour  at  50-55°,  with  stirring.  It  is  then  strained  through 
muslin,  yielding  a  filtrate  of  deep  red  color,  which  should  be  made 
to  equal  1000  c.c.  This  filtrate  is  placed  in  the  inner  compart- 
ment of  a  double  boiler  (rice  cooker)  and  to  it  are  added  10  grams 
peptone,  5  grams  sodium  chloride  and  100  to  150  grams  of  sheet 
gelatin  of  the  best  quality  ("gold  label"  gelatin).  The  larger 
amount  of  gelatin  should  be  used  during  warm  weather  if  no  low- 
temperature  incubator  is  at  hand.  These  constituents  are  dissolved 
by  stirring  at  a  temperature  below  55°  C,  After  complete  solution, 
the  reaction  is  titrated  as  has  been  described  for  the  titration  of 
broth.  From  30  to  50  c.c.  of  normal  alkali  are  usually  required 
to  give  the  proper  reaction  to  a  liter  of  the  medium.  After  this 
has  been  ascertained,  and  the  amount  added,  the  medium  is 
thoroughly  mixed  and  then  left  covered  and  undisturbed  while 
the  water  in  the  outer  compartment  of  the  cooker  is  boiled  for 
an  hour.  It  is  well  to  have  boiling  water  at  hand  in  another 
receptacle  so  that  the  supply  in  the  cooker  may  be  replenished 
if  it  gets  low,  without  chilling  the  medium.  The  gelatin  is  now 
filtered  through  paper  wet  with  hot  water,  and  should  be  kept 
warm  during  filtration  by  means  of  a  funnel-heater,  or  by  a  steam 


CULTURE    MEDIA 


89 


bath,  although  these  are  not  essential.  If  it  gets  cold  it  may  be 
poured  out  of  the  funnel  and  warmed  again  in  the  pan.  A  portion 
of  the  nitrate  should  be  boiled  in  a  test  tube  over  the  flame  for  a 
minute  or  two.  It  should  then  remain  (i)  perfectly  clear,  (2) 
alkaline  to  litmus  paper,  and  (3)  should  solidify  on  cooling  in  tap 
water.  After  nitration  the  medium  is  filled  into  tubes  and  steril- 
ized in  streaming  steam  by  the  fractional  method,  20  minutes  at 
1 00°  C.  for  3  consecutive  days.  Gelatin 
may  be  sterilized  in  the  autoclave  at  110°  C. 
for  10  minutes,  but  it  should  be  chilled  in 
cold  water  at  once  after  removal,  and  even 
then  its  gelatinizing  property  may  be  seri- 
ously impaired. 

In  filling  gelatin  into  tubes  it  is  important 
that  the  medium  should  not  be  spilled  on 
the  mouth  of  the  tube  or  on  the  cotton  plug, 
as  this  accident  causes  the  latter  to  be  glued 
in  position.  The  filling  apparatus  indicated 
in  Fig.  33  will  be  found  convenient  for  filling 
any  sort  of  liquid  medium  into  tubes,  and 
with  proper  care  one  may  fill  tubes  rapidly 
without  soiling  the  mouths  of  tubes  and  their 

cotton  plugs.  Fio.33.-Apparatusfor 

Gelatin  may  be  made  from  beef  extract,    filling  media  into  tubes. 

mi  I.L         j        i    j.'  It  is  held  in  a  ring-stand 

The  extract,   peptone,  salt  and  gelatin  are    support. 
dissolved  at  a  temperature  below  60°  C.  or 
the  medium  is  cooled  to  this  temperature  after  solution  has  been 
accomplished.     It  is   titrated  and  the  proper  amount  of   alkali 
added.     An  egg  is  beaten  up  in  water  and  then  stirred  into  the 
medium.     It    is    then  boiled  on  the  water  bath  for  an  hour, 
filtered,  tested,  tubed  and  sterilized. 

Nutrient  Agar. — To  a  liter  of  nutrient  broth,  prepared  as 
above  described  (page  84)  add  15  grams  of  finely  cut  agar 
shreds.  Weigh  the  pan  with  its  contents.  Boil  the  material 
over  the  direct  flame  for  one  to  two  hours,  with  constant  stirring 


QO  BACTERIOLOGY 

to  avoid  burning,  adding  hot  distilled  water  from  time  to  time 
to  compensate  for  the  loss  by  evaporation.  Instead  of  boiling 
it  is  convenient  to  cook  the  medium  in  the  autoclave  at  110°  C. 
for  45  minutes  to  an  hour.  In  either  case,  the  agar  should  be 
very  completely  dissolved.  The  medium  is  then  cooled  to  60°  C. 
and  an  egg  previously  beaten  up  in  water  is  added  and  thor- 
oughly mixed  with  the  agar.  It  is  then  boiled  again  for  10  minutes 
over  the  free  flame,  with  constant  stirring  at  the  bottom,  or  for 
45  minutes  on  the  water  bath,  or  for  15  minutes  in  the  auto- 
clave at  110°  C.  Distilled  water  is  added  to  restore  the  original 
weight,  and  the  medium  is  then  filtered,  usually  through  a  layer 
of  cotton  wet  with  hot  water,  although  filter  paper  may  be  used. 
Filtration  is  favored  by  keeping  the  funnel  hot,  either  with  the 
hot-water  funnel  heater  or  in  a  steam  bath,  and  it  may  be  hastened 
by  the  use  of  suction.  The  filtrate  need  not  be  perfectly  clear, 
and  it  usually  clouds  on  cooling  unless  it  is  acid  in  reaction.  The 
reaction  should  be  alkaline  to  litmus.  After  filling  into  tubes  or 
flasks,  agar  should  be  sterilized  in  the  autoclave  at  110°  C.  for  30 
to  35  minutes. 

Modifications  of  the  Common  Media.— Broth  is  made  nearly 
free  from  sugar  by  fermenting  the  meat  infusion  over  night  at 
37°  C.  after  inoculating  it  with  B.  coli,  and  then  proceding  with 
the  filtrate  in  the  usual  way.  This  medium  is  designated  as 
sugar-free  broth.  Various  sugars  or  other  substances  are  added 
to  such  broth  in  order  to  test  the  ability  of  bacteria  to  ferment 
them.  Acetic  acid,  0.5  per  cent,  is  added  to  broth  to  make  a 
selective  medium  for  acid-resisting  bacteria.  Glycerin,  5  to  7 
per  cent,  is  added  to  broth  for  the  cultivation  of  the  tubercle 
bacillus.  Naturally  sterile  ascitic  fluid  or  blood  is  added  to  broth 
to  promote  the  growth  of  certain  types  of  microbes,  and  to  en- 
courage anaerobes.  Bits  of  naturally  sterile  tissue  are  added 
to  broth  for  similar  purposes. 

Gelatin  is  modified  by  the  addition  of  various  sugars,  especially ' 
dextrose  and  lactose,  often  with  the  further  addition  of  litmus. 
The  production  of  acid  by  fermentation  of  the  sugar  is  at  once 


CULTURE    MEDIA  9 1 

evidenced  by  the  reddening  of  the  litmus.  Glucose  litmus  gela- 
tin is  also  a  useful  medium  for  anaerobes.  It  is  best  to  sterilize, 
the  litmus  separately  and  add  it  from  a  sterile  pipette  at  the  time 
the  medium  is  used. 

Agar  is  modified  by  the  addition  of  5  to  7  per  cent  of  glycerin, 
and  such  glycerin-agar  is  used  extensively  for  cultivation  of  the 
tubercle  bacillus  and  several  other  pathogenic  bacteria.  Various 
sugars,  supplemented  by  the  addition  of  litmus,  are 
dissolved  in  agar  to  test  the  fermentation  properties 
of  bacteria.  Glucose  agar  is  extensively  employed  as 
such  for  the  cultivation  of  anaerobes.  Agar  also  forms 
the  gelatinizing  base  for  a  number  of  more  or  less  com- 
plex special  media. 

STERILIZABLE  SPECIAL  MEDIA. 

Potato. — Potatoes  were  perhaps  the  first  solid  med- 
ium employed  in  the  cultivation  of  micro-organisms. 
Boiled  or  steamed  potatoes  kept  in  a  moist  place,  such 
as  a  large  covered  glass  dish,  may  well  be  employed  as 
an  illustration  of  primitive  technic,  and  excellent  cul- 
tures of  the  common  chromogenic  bacteria  may  be  ob- 
tained in  this  way.     For  most  purposes  it  is  better  to 
put  pieces  of  potato  in  test-tubes  where  they  are  more 
perfectly  protected  from  contamination,  as  suggested 
by  Bolton.1     The  potato  is  carefully  washed,  a  slice    Potato4  in 
removed  from  each  end,  and  a  cylinder  is  cut  out  with    culture 
a  cork-borer  or  with  a  test  tube  cut  off  near  its  bottom. 
This  cylinder  is  divided  diagonally  into  two  pieces.     The  pieces 
are  washed  in  running  water  for  twelve  to  eighteen  hours. 
They  are  placed  in  test-tubes  containing  a  little  water  to  keep 
the  potato  moist,  and  are  supported  from  the  bottom  on  a  piece 
of  glass  tubing  about  i  to  2  cm.  in  length  (or  on  cotton,  or  in  a 
specially  devised  form  of  tube  with  a  constriction  at  the  bottom) . 

1  Bolton,  The  Medical  News,  Vol.  I,  1887,  p.  318. 


g  2  BACTERIOLOGY 

The  tubes  are  plugged,  and  sterilized  in  the  autoclave  at  110°  C. 
for  30  minutes.  Potato  is  best  when  freshly  prepared;  it  is  likely 
to  become  dry  and  discolored  with  keeping. 

Milk. — Milk  fresh  as  possible  is  placed  in  a  covered  jar, 
steamed  for  fifteen  minutes,  and  then  kept  on  ice  for  twenty- 
four  hours.  At  the  end  of  that  time  the  middle  portion  is  re- 
moved by  means  of  a  siphon.  The  upper  and  lower  layers  must 
not  be  taken;  the  upper  part  contains  cream,  and  the  lower  part 
particles  of  dirt,  both  of  which  are  to  be  avoided.  About  7  to  10 
c.c.  are  to  be  run  into  each  test  tube.  The  tube  is  plugged  with 
cotton,  and  sterilized  in  the  autoclave  at  110°  C.  for  30  minutes. 

The  coagulation  of  milk,  which  is  accomplished  by  certain 
bacteria,  is  a  very  valuable  differential  point.  A  little  litmus 
tincture  may  be  added  to  the  tubes  of  milk  before  sterilizing, 
until  they  acquire  a  blue  color,  to  indicate  whether  or  not  acids 
are  formed  by  the  bacteria  which  are  afterward  cultivated  in 
the  milk. 

Dunham's  Peptone  Solution. 

Peptone 10  grams. 

Sodium  chloride 5  grams. 

Water i  liter. 

Boil,  filter,  sterilize  in  the  usual  manner. 

Dunham's  solution  is  valuable  to-  test  the  development  of 
indol  by  bacteria  (see  Part  II.,  Chapter  VIII.).  The  develop- 
ment of  acids  may  be  detected  after  the  addition  of  2  per  cent  of 
rosolic  acid  solution  (0.5  per  cent  solution  in  alcohol);  alkaline 
solutions  give  a  clear  rose-color  which  disappears  in  the  presence 
of  acids. 

Nitrate  Broth. — Dissolve  i  gram  of  peptone  in  1000  c.c.  of 
distilled  water,  and  add  2  grams  of  nitrite-free  potassium  nitrate. 
Fill  into  test-tubes,  10  c.c.  in  each,  and  sterilize  in  the  autoclave 
at  110°  C.  for  15  minutes. 

Blood -serum. — The  blood  of  the  ox  or  cow  may  be  obtained 
easily  at  the  abattoir.  It  should  be  collected  in  a  clean  jar. 
When  it  has  coagulated,  the  clot  should  be  separated  from  the 


CULTURE   MEDIA  93 

sides  of  the  jar  with  a  glass  rod.  It  may  be  left  on  the  ice  for 
from  twenty-four  to  forty-eight  hours.  At  the  end  of  that  time 
the  serum  will  have  separated  from  the  clot  and  may  be  drawn 
off  with  a  siphon  into  tubes.  These  tubes  are  sterilized  for  the 
first  time  in  a  slanting  position,  as  the  first  sterilization  coagulates 
the  serum.  The  coagulation  may  be  done  advantageously,  as 
advised  by  Councilman  and  Mallory,  in  the  hot-air  sterilizer  at  a 
temperature  below  the  boiling-point.  After  coagulation,  sterilize 
in  the  autoclave  at  110°  C.  for  20  minutes.  This  serum  makes  an 
opaque  medium  of  a  cream  color.  Blood-serum  may  be  more 


FIG.  35. — Koch's  serum  sterilizer. 

conveniently  sterilized  in  the  Koch  serum  inspissator  (Fig.  35). 
A  clear  blood-serum  is  to  be  obtained  by  sterilization  at  a  tempera- 
ture of  58°  C.  for  one  hour,  on  each  of  six  days,  if  a  fluid  medium 
is  desired,  or  of  75°  C.  on  each  of  four  days  if  the  serum  is  to  be 
solidified.  In  the  latter  case  the  tubes  are  to  be  placed  in  an  in- 
clined position.  Opaque,  coagulated  blood-serum  has  most  of 
the  advantages  of  the  clear  medium.  Blood-serum  may  be  se- 
cured from  small  animals  by  collecting  blood  directly  from  the 
vessels,  and  with  proper  technic  may  be  obtained  in  a  sterile 
condition;  and  the  serum  may  be  separated  and  stored  in  a  fluid 
state.  Human  blood-serum  is  sometimes  obtained  from  the 


94  BACTERIOLOGY 

placental  blood.  The  preservation  of  blood-serum  is  sometimes 
accomplished  with  chloroform,  of  which  i  per  cent  is  to  be  added 
to  the  medium;  in  this  manner  the  serum  may  be  preserved  for  a 
long  time.  It  may  be  filled  into  tubes,  solidified  and  sterilized 
as  required;  the  chloroform  will  be  driven  off  by  the  heat,  owing 
to  its  volatility.  Blood-serum  media  which  are  sterilized  at 
low  temperatures  should  be  tested  for  twenty-four  hours  in  the 
incubator  to  prove  that  sterilization  has  been  effective;  if  it  has 
not,  development  of  the  contaminating  bacteria  will  take  place 
and  be  visible  to  the  eye. 

Loffler's  blood-serum  consists  of  one  part  of  bouillon  con- 
taining i  per  cent  of  glucose,  mixed  with  three  parts  of  blood- 
serum.  It  is  sterilized  like  ordinary  blood-serum.  It  is  used 
largely  for  the  cultivation  of  the  bacillus  of  diphtheria. 

Fresh  eggs  in  their  shells  may  be  used  without  other  preparation 
than  washing  the  surface  thoroughly  with  bichloride  of  mercury 
solution;  or  after  sterilization  by  steam,  which  of  course  coagu- 
lates the  albumen.  The  egg  is  easily  inoculated  through  a  small 
opening  made  with  a  heated  needle,  which  may  be  closed  after- 
ward with  collodion.  Hueppe  recommended  eggs  closed  in  this 
manner  for  the  cultivation  of  anaerobic  bacteria. 

Dorset's  Egg  Medium.1 — Perfectly  fresh  eggs  are  washed  and 
the  shells  sterilized  with  bichloride  solution.  The  eggs  are  then 
carefully  broken  and  the  yolks  and  whites  mixed  in  a  sterile 
dish.  The  mixed  material  is  poured  into  sterile  tubes  and  solidi- 
fied in  the  slanting  position  by  heating  at  70 — 75°  C.  for  two 
hours.  Contamination  with  bacteria  should  be  carefully  avoided 
throughout  the  preparation  of  the  medium.  The  tubes  should 
be  sealed  with  rubber  caps  or  with  wax  and  incubated  for  a 
week  before  use.  It  is  well  to  moisten  the  surface  with  a  few 
drops  of  sterile  water  from  a  pipette  before  inoculating  the  me- 
dium. This  medium  is  used  for  growing  the  tubercle  bacillus. 

Bread-paste. — Dry  or  toasted  bread  is  broken  into  small 
crumbs,  filled  into  tubes  or  flasks,  moistened  with  water  and 

1  Dorset:  American  Medicine,  April  5,  1902. 


CULTURE   MEDIA 


95 


sterilized  in  the  autoclave.     This  medium  is  used  for  cultivation 
of  molds. 

MEDIA  CONTAINING  UNCOOKED  PROTEIN. 

Culture  media  containing  naturally  sterile  uncooked  protein 
have  made  possible  the  cultivation 
of  microbic  forms  not  cultivable  on 
other  media.  Many  microbes 
which  may  also  grow  on  cooked 
media  do  much  better  on  those  con- 
taining uncooked  protein.  It  would 
seem  that  media  of  this  kind  are  to 
play  an  important  part  in  the  fur- 
ther development  of  our  knowledge 
of  pathogenic  micro-organisms. 

Collection  of  Sterile  Blood. — A 
few  drops  of  blood  may  be  obtained 
from  the  ear  lobe.  The  skin  is 
cleaned  with  soap  and  alcohol  and 
then  dried  perfectly  with  sterile 
cotton.  It  is  punctured  with  a  ster- 
ilized lancet  and  the  blood  quickly 
transferred  to  the  surface  of  an  agar 
slant  by  means  of  a  platinum  loop 
or  a  sterile  capillary  pipette.  It 
should  be  incubated  before  use  to 
insure  sterility. 

Larger     quantities     of     sterile 
human  blood  may  be  obtained  with 
far    less   danger   of   contamination 
from   the  median    basilic   vein 
other    large    vein    at    the 
i  The    skin    is   washed,    disinfected 

with  alcohol  and  bichloride  and  dried.     An  elastic  bandage  is 
applied  about  the  arm  to  distend  the  veins.     A  sterile  needle 


FIG..  36. — Pipette  with  needle  at- 
tached   for    drawing    human    blood 
or   from  a  vein  for  use  in  culture  media. 
The  glass  rod  inside  is  used  to  defi- 
elbow.   brinate  the  blood. 


96  BACTERIOLOGY 

attached  to  a  special  sterilized  blood  pipette  is  thrust  into 
the  vein  and  the  desired  amount  of  blood  collected  (see 
Fig.  36).  It  may  be  allowed  to  clot  if  sterile  serum  is  desired, 
or  it  may  be  defibrinated  by  stirring  with  the  glass  rod  if  a  mix- 
ture of  corpuscles  and  serum  is  desired,  or  it 
may  be  kept  in  the  fluid  state  by  the  addi- 
tion of  sterile  10  per  cent  solution  of  sodium 
citrate  so  that  the  final  mixture  may  con- 
tain i  per  cent  of  citrate.  The  bandage  is 
removed  from  the  arm  before  the  needle  is 
withdrawn.  Pressure  over  the  wound  with 
cotton  wet  in  alcohol  for  five  minutes  pre- 
vents subcutaneous  hemorrhage.  No  dress- 
ing is  required.  The  inlet  to  the  blood  pip- 
ette is  closed  by  kinking  the  rubber  tube. 
The  blood  or  the  serum  is  subsequently 
handled  by  means  of  sterilized  pipettes,  and 
most  conveniently  by  means  of  the  Pasteur 
bulb  pipettes.  (See  page  33.) 

Blood  from  small  laboratory  animals 
serves  as  well  as  human  blood  for  most  pur- 
poses. It  may  be  drawn  from  the  carotid 
artery  by  aseptic  technic  into  a  special  blood 
pipette  the  lower  end  of  which  is  drawn  out 

FIG.   37—  Pipette    into  a  capillary  which  is  inserted   directly 
with    capillary    tip    for     .  .  ,  . 

drawing  blood  fromcaro-    into  the  artery   (see  Fig.  37).     This  blood 


WtoNw:)  an  anima1'    may  be  defibrinated,  citrated  or  allowed  to 

clot. 

Small  amounts  of  sterile  blood  may  be  obtained  from  labora- 
tory animals  without  killing  them  by  means  of  heart  puncture. 
The  needle  of  a  Luer  glass  syringe  is  inserted  through  the  chest 
wall,  after  anesthetizing  the  animal  and  shaving  and  disinfecting 
the  skin,  so  as  to  enter  the  cavity  of  the  right  ventricle.  A 
quantity  of  blood  not  greater  than  yV  the  weight'  of  the  animal 
may  be  removed.  The  needle  is  withdrawn  and  the  blood  quickly 


CULTURE    MEDIA  97 

forced  out  into  a  sterile  tube  where  it  may  be  defibrinated 
or  mixed  with  citrate  solution,  or  allowed  to  clot,  as  may  be- 
desired. 

Very  large  amounts  of  sterile  blood  are  best  obtained  from 
the  jugular  vein  of  the  horse  or  the  superficial  abdominal  veins 
of  the  cow.  The  skin  is  shaved,  washed  and  cauterized  with  95 
per  cent  carbolic  acid.  When  this  has  dried  the  vein  is  punctured 
with  the  needle,  which  is  attached  to  a  suitable  glass  receptacle 
by  means  of  rubber  tubing. 

Collection  of  Sterile  Ascitic  Fluid.— For  this  purpose  a  large 
trochar  and  canula  provided  with  a  lateral  outlet,  and  made  so 
that  the  trochar  can  be  drawn  back  beyond  this  outlet  without 
being  completely  removed,  is  most  convenient.  The  instrument 
is  oiled  with  liquid  paraffin.  A  rubber  tube  about  40  cm.  in 
length  is  attached  to  the  outlet  and  the  whole  is  wrapped  in  a 
cloth  and  sterilized  in  the  autoclave.  The  site  selected  for  punc- 
ture should  be  cleansed  and  painted  with  tincture  of  iodine  and 
the  skin  may  be  frozen  with  ethyl  chloride  if  desired.  One  man 
inserts  the  trochar  and  canula,  taking  care  not  to  contaminate 
it  after  it  is  removed  from  the  cover.  Another  manipulates  the 
attached  rubber  tube,  carefully  guarding  it  from  contamination 
and  allowing  the  fluid  to  flow  into  sterilized  flasks  of  1000  c.c. 
capacity  which  are  handled  by  an  assistant.  The  mouth  of  each 
flask  should^be  flamed  after  removing  the  cotton  plug  and  again 
before  it  is  inserted  after  filling  the  flask.  With  proper  technic 
the  ascitic  fluid  will  as  a  rale  be  found  bacteria-free.  It  should 
be  stored  in  a  cool  place,  and  is  most  conveniently  handled  by 
means  of  large  Pasteur  bulb  pipettes. 

In  collecting  hydrocele  fluid  or  other  fluids  to  be  used  for 
culture  media,  similar  aseptic  technic  should  be  employed. 

Sterilization  of  Contaminated  Fluids. — Any  of  the  clear  fluids 
may  be  sterilized,  when  this  is  necessary,  by  filtration  through  the 
Berkefeld  filter.  The  filtrate  will  usually  prove  less  valuable 
as  a  medium  than  the  corresponding  unfiltered  naturally  sterile 
material. 


98  BACTERIOLOGY 

Collection  of  Sterile  Tissue. — For  this  purpose,  a  healthy 
animal  is  first  bled  to  death  as  described  above  (page  96)  for  the 
collection  of  sterile  blood.  The  skin  is  then  thoroughly  wet  with 
water  or  with  bichloride  solution.  With  sterile  instruments,  an 
incision  is  made  in  the  median  line  and  the  skin  carefully  stripped 
back.  It  is  then  well  to  sear  the  abdominal  wall  with  a  hot  iron 
along  the  median  line  and  also  crosswise  and  cut  along  these 
lines  with  sterile  scissors,  opening  the  abdominal  cavity.  The 
organs  desired  are  quickly  removed  with  sterile  instruments 
and  placed  in  covered  sterile  glass  dishes.  The  liver,  kid- 
neys and  testes  are  the  organs  most  frequently  employed  in 
culture  media.  They  are  divided  into  pieces  of  suitable  size 
with  sterile  scissors.  Brain  tissue  may  be  readily  obtained  from 
the  rabbit.  The  top  of  the  head  is  skinned  and  an  opening 
made  by  cutting  away  the  skull  between  the  orbits  with  the  bone 
forceps.  An  area  of  the  anterior  portion  of  the  brain  is  exposed. 
This  is  thoroughly  seared  with  a  hot  iron,  as  well  as  the  adjacent 
structures.  A  Pasteur  bulb  with  a  large  capillary  (internal 
diameter  at  least  5  mm.)  is  convenient  for  drawing  out  the 
brain  tissue.  This  large  capillary  is  inserted  through  the  seared 
area  and  the  brain  is  broken  up  by  moving  it  about  in  the 
cranial  cavity,  while  the  tissue  is  drawn  into  the  bulb  by  suction. 

Pfeiffer's  Blood-streaked  Agar. — A  large  loopful  of  naturally 
sterile  human  blood,  freshly  taken  from  the  ear,  is  spread  over 
the  surface  of  an  agar  slant,  and  incubated  to  insure  sterility. 
This  medium  is  employed  for  cultivation  of  the  influenza 
bacillus. 

Novy's  Blood-agar. — The  agar  is  melted  and  cooled  to  50° 
C.  The  naturally  sterile  defibrinated  blood,  usually  rabbit's 
blood,  is  warmed  to  about  40°  C.  The  blood  is  mixed  with  the 
agar  in  various  proportions,  and  the  mixture  is  allowed  to  solidify 
in  the  inclined  position.  The  medium  should  be  fairly  firm  in 
consistency  and  some  fluid  should  collect  at  the  bottom  of  the 
slant.  The  medium  is  useful  for  cultivation  of  the  gonococcus, 
the  influenza  bacillus,  streptococcus,  pneumococcus  and  meningo- 


CULTURE   MEDIA  99 

coccus,  but  more  especially  for  cultivation  of  the  flagellated 
hematozoa  such  as  trypanosomes  and  related  organisms,  including 
the  Leishman-Donovan  bodies. 

Smith's  Broth  Containing  Sterile  Tissue. — Pieces  of  naturally 
sterile  organs,  usually  liver  or  kidney,  are  placed  in  broth,  more 
particularly  in  fermentation  tubes  of  broth.  The  bits  of  tissue 
are  conveniently  handled  by  touching  with  a  hot  platinum  wire 
or  glass  capillary,  to  which  they  will  adhere.  The  medium  is 
especially  useful  for  the  culture  of  anaerobic  bacteria.  Naturally 
sterile  blood  added  to  the  broth  also  serves  for  this  purpose. 

Ascitic-fluid-agar. — This  is  made  in  the  same  way  as  the 
Novy's  blood-agar  except  that  naturally  sterile  human  ascitic 
fluid  is  employed  instead  of  blood.  The  medium  is  beautifully 
transparent,  and  may  be  employed  for  plating  as  well  as  for  tube 
cultures.  It  is  especially  valuable  for  cultivation  of  the  gono- 
coccus  and  also  for  the  streptococcus,  pneumococcus  and 
meningococcus. 

Noguchi's1  Ascitic  Fluid  with  Sterile  Tissue. — Naturally 
sterile  tissue  is  placed  in  a  tall  tube.  A  deep  layer  of  ascitic  fluid 
is  added,  and  for  some  purposes  this  is  covered  with  a  layer  of 
sterile  paraffin  oil.  The  medium  is  used  more  especially  for  the 
cultivation  of  the  blood  spirochetes  which  cause  relapsing  fever. 

1  Noguchi:  Journ.  Exp.  Med.,  Jan.  i,  1912,   Vol.  XV,  pp.  90-100. 


CHAPTER     IV. 

COLLECTION  OF  MATERIAL  FOR  BACTERIOLOGICAL 

STUDY. 

Bacteria  under  natural  conditions  are  usually  associated  as 
mixtures  of  several  species  living  together.  Only  under  rather 
exceptional  circumstances  will  a  single  kind  of  bacteria  be  found 
growing  alone.  This  does  occur  in  disease,  however,  where  the 
living  host  may  be  able  to  keep  out  all  but  the  one  kind  of  mi- 
crobe. But  even  diseased  tissues  or  exudates  originally  harbor- 
ing only  one  kind  of  bacteria  may  quickly  acquire  others  in  abun- 
dance after  removal  from  the  living  body.  It  is  well  therefore 
to  regard  any  material  presented  for  bacteriological  examination 
as  potentially,  and  in  all  probability  actually,  harboring  several 
kinds  or  species  of  bacteria.  The  direct  planting  of  such  material 
on  a  culture  medium  .will,  therefore,  in  most  instances  give  rise 
to  a  mixed  culture,  in  which  those  forms  least  prominent  in  the 
original  material  may  easily  appear  as  most  important.  If  the 
material  be  allowed  to  stand,  especially  if  it  be  a  favorable 
medium  for  bacterial  growth,  the  relationships  present  may  be- 
come seriously  confused.  It  should,  therefore,  be  examined  as 
fresh  as  possible.  When  immediate  examination  is  impossible 
the  material  should  be  kept  on  ice. 

Samples  of  water,  milk  or  other  fluid  should  be  collected  in 
sterilized  tubes  or  bottles.  Samples  of  solid  food  should  be 
seared  or  charred  all  over  the  surface  and  divided  with  a  sterilized 
knife.  A  small  piece  of  the  interior  is  then  removed  to  a  sterilized 
glass  dish  and  covered. 

Material  removed  from  the  human  or  from  the  animal  body  dur- 
ing life  or  at  autopsy  may.be,  bacteria-free, :  or  it  may  contain 
one  or  more  specie^,  of  -microbes.  „  Ijt  is  irnportaiit  that  the  picture 

100 


MATERIAL   FOR  BACTERIOLOGICAL    STUDY  IOI 

be  not  confused  by  the  addition  of  bacteria  from  the  surface  of 
the  body,  from  instruments  or  from  the  air  during  the  collection^ 
and  transportation  to  the  laboratory.  Unfortunately  the 
laboratory  study  of  such  material  is  too  often  rendered  untrust- 
worthy or  worthless  through  lack  of  attention  to  this  point. 

When  merely  microscopic  examination  is  to  be  undertaken, 
contamination  may  not  be  serious,  and  an  antiseptic,  such  as 
two  per  cent  of  carbolic  acid,  may  be  added  to  the  material,  if 
fluid,  and  if  solid  it  may  be  immersed  in  ten  per  cent  formalin. 
The  bottles  used  should  be  new  and  clean.  Such  material  may 
also  be  spread  on  microscopic  slides  or  cover-glasses  in  a  thin 
layer,  dried,  fixed  in  the  flame,  and  transported  to  the  labora- 
tory. This  method  is  not  always  free  from  danger  when  the 
material  passes  through  several  hands.  Special  precautions  for 
collecting  material  for  microscopic  examination  will  be  considered 
in  discussing  the  specific  pathogenic  microbes. 

Specimens  of  sputum  should  be  raised  from  the  trachea,  bron- 
chi and  lungs  after  previously  cleansing  the  mouth.  Sputum 
should  be  received  into  a  sterile  wide-mouthed  bottle,  and  stop- 
pered with  a  sterilized  cork.  The  exterior  of  the  bottle  should 
then  be  carefully  washed  with  5  per  cent  carbolic  acid. 

Urine  should  be  collected  by  catheter  with  careful  aseptic 
technic,  and  should  be  received  in  a  clean  sterilized  bottle. 

Blood  and  transudates  are  collected  by  the  technic  previously 
described  (page  95).  Blood  is  drawn  from  the  vein  by  means 
of  the  Luer  syringe  and  is  quickly  ejected  into  several  flasks  of 
broth  (150  to  250  c.c.)  and  into  Petri  dishes  where  it  is  mixed  with 
melted  agar,  (cooled  to  50°  C.  )  before  clotting  takes  place. 

Cerebro-spinal  fluid  is  obtained  by  inserting  a  sterilized  needle 
(4  cm.  long  for  children,  8-10  cm.  long  for  adults,  and  with 
lumen  i  mm.)  a  little  to  one  side  of  the  median  line  in  the  back, 
so  that  it  enters  the  spinal  canal  between  the  second  and  third, 
or  between  the  third  and  fourth,  lumbar  vertebrae.  Aseptic 
technic  is  essential.  The  fluid  coming  from  the  needle  is 
received  in  a  sterile  tube. 


102 


BACTERIOLOGY 


Feces  from  infants  and  young  children  are  best  collected  by 
means  of  a  heavy  glass  tube  closed  and  rounded  off  at  the  end,  and 
provided  with  a  lateral  opening  near  the  closed  end.  This  is 
enclosed  in  a  larger  tube  and  sterilized.  It  is  inserted  well  into 
the  rectum  with  aseptic  technic  and  the  entrance  of  fecal  material 
through  the  lateral  opening  is  favored  by 
gently  moving  the  tube.  It  is  then  with- 
drawn and  replaced  in  its  original  container 
to  be  transported  to  the  laboratory.  From 
adults  the  feces  are  passed  directly  into  a 
sterilized  covered  agateware  basin  without 
other  special  apparatus. 

Intestinal  juice  from  the  duodenum  may 
be  obtained  in  infants1  by  inserting  a  sterile 
rubber  catheter,  closed  below  with  a  steri- 
lized gelatin  capsule,  through  the  esophagus 
and  stomach  into  the  duodenum.  The  cap- 
sule is  then  blown  off  by  pressure  from  a 
sterile  syringe  attached  at  the  other  end  of 
the  catheter  and  the  fluid  contents  of  the 
duodenum  aspirated.  In  adults2  the  Einhorn 
duodenal  tube  is  employed.  The  tube  is 
of  instrument  for  obtain-  sterilized  by  boiling  and  the  lower  opening 

mg  feces  from  infants  for  J 

bacteriological  examina-  sealed  with  a  sterilized  gelatin  capsule  and 
by  finally  coating  with  shellac.  The  tube  is 
inserted  through  the  esophagus  and  is  carried 
through  the  pylorus  by  peristalsis.  Ordinarily  it  is  inserted  in 
the  evening.  On  the  following  morning  the  seal  at  the  lower  end 
is  broken  by  pressure  of  a  sterile  syringe  attached  to  the  free  end 
of  the  tube  and  the  sample  of  juice  aspirated.  Intestinal  juice 
may  be  obtained  from  various  levels  in  the  jejunum  also  by  regu- 
lating the  length  of  tube  inserted. 

Pus  and  other  exudates  are  best  collected  in  sterile  glass  capil- 


FIG.  38. — Two  types 


tion.    (After  Schmidt  and 
Strasburger.) 


1Hess:  Journ.  Infections  Diseases,  July  1912,  Vol.  XI,  pp.  71-76- 
2MacNeal  and  Chace:    Arch.  Int.  Med.,  Aug.,  1913,  Vol.  XII,  pp.  178-197- 


MATERIAL   FOR  BACTERIOLOGICAL    STUDY  103 

lary  pipettes   (see  page  33).     A  sterilized    cotton    swab,   made 
by  winding  a  pledget  of  absorbent  cotton  around  the  end  of  a  stiff- 
wire,  enclosing  it  in  a  test-tube  and  sterilizing  it,  is  also  useful, 
especially  when  it  is  impossible  or  undesirable  to  employ  the 
glass  tube. 

At  autopsies  on  human  subjects,  the  same  principles  for  col- 
lection of  material  apply.  Fluids  are  best  collected  in  sterile 
glass  pipettes  and  even  solid  organs  may  be  seared  and  punctured 
with  a  strong  glass  capillary  into  which  some  of  the  pulp  is  drawn 
by  suction.  The  tubes  may  be  sealed  in  the  flame  and  trans- 
ported considerable  distances  to  the  laboratory.  This  is  usually 
more  satisfactory  than  the  inoculation  of  culture  media  in  the 
autopsy  room,  especially  if  the  facilities  for  bacteriological  work 
there  are  somewhat  limited.  Smears  on  slides  or  cover-glasses 
should  also  be  made  for  microscopic  examination,  and  pieces  of 
the  various  organs  fixed  in  alcohol  or  formalin  and  preserved 
for  sectioning. 


CHAPTER  V. 
THE  CULTIVATION  OF  MICRO-ORGANISMS. 

Avoidance  of  Contamination. — Micro-organisms  are  so  numer- 
ous on  the  body  of  man  and  in  his  environment  that  they  are  likely 
to  be  present  on  all  articles  about  us  unless  special  precautions 
are  taken  to  remove  or  destroy  them.  The  dust  blown  about 
in  the  air  contains  bacteria  and  spores  of  molds.  The  primary 
essential  in  all  bacteriological  culture  work  is  the  exclusion  of 
these  extraneous  micro-organisms.  The  unskilled  or  careless 
worker  may  quickly  add  some  of  these  chance  organisms  to  the 
material  which  he  is  attempting  to  study,  introducing  an  element 
of  almost  hopeless  confusion  unless  it  is  recognized.  Another 
essential  of  great  importance,  especially  when  working  with  patho- 
genic microbes,  is  the  complete  destruction  of  all  living  bacteria 
before  they  are  allowed  to  pass  beyond  strict  and  absolute  con- 
trol. The  unskilled  or  careless  worker  in  the  laboratory,  who 
allows  micro-organisms  to  escape  from  him  while  he  is  attempt- 
ing to  study  them,  is  a  serious  menace  not  only  to  himself  but  to 
all  others  in  the  laboratory.  These  two  primary  essentials  must 
be  mastered  by  practice  in  handling  harmless  forms. 

Every  instrument  with  which  bacteria  are  handled  should  be 
sterilized  before  it  is  used,  and  again  after  use.  In  the  case  of 
the  commonly  used  platinum  wire,  this  sterilization  is  accom- 
plished in  the  flame.  The  wire  is  heated  to  a  glow  and  allowed 
to  cool  before  handling  bacteria,  and  immediately  after  its  use, 
before  it  leaves  the  hand,  it  is  brought  close  to  the  flame  so  as  to 
dry  the  material  on  it  and  then  again  heated  to  redness.  Care- 
ful drying  in  this  way  avoids  sputtering  and  consequent  scattering 
of  bacteria,  which  is  almost  certain  to  occur  if  moist  material, 
especially  fat  or  protein,  is  immediately  thrust  into  the  flame. 

104 


THE    CULTIVATION    OF   MICRO-ORGANISMS  1 05 

In  using  the  Bunsen  flame  for  sterilization,  the  innermost  cone 
near  the  base  of  the  flame  may  be  utilized  for  drying  material  on_ 
the  end  of  the  wire.  This  inner  cone  is  not  burning  and  is  com- 
paratively cool,  and  after  a  little  practice  the  end  of  the  wire 
is  easily  brought  into  it  and  dried  without  sputtering.  Slowly 
elevating  the  wire  brings  it  gradually  into  hotter  zones  of  the 
flame  until  it  glows. 

Bacteria  do  not  of  themselves  leave  a  moist  surface.  They 
are  not  even  removed  by  moderate  currents  of  air  unless  they 
have  been  previously  dried.  Their  distribution  about  the  labora- 
tory, therefore,  results  from  relatively  gross  accidents  or 
gross  carelessness.  When  material  containing  bacteria  is  acci- 
dentally spilled,  it  should  be  covered  at  once  with  disinfectant 
solution,  such  as  i-iooo  mercuric-chloride  solution.  As  a  rou- 
tine procedure  it  is  well  to  wash  the  work  table  daily  with  bi- 
chloride solution  and,  when  working  with  pathogenic  bacteria, 
to  wash  the  hands  at  the  end  of  the  day's  work,  first  with  the 
bichloride  solution  and  then  with  soap  and  water. 

Isolation  of  Bacteria. — In  order  to  study  any  kind  of  bacteria 
it  is  necessary  to  have  the  particular  species  separated  from  other 
sorts  with  which  it  may  be  mixed.  The  earlier  bacteriologists 
endeavored  to  separate  bacteria  of  different  sorts  by  successive 
transplantations  through  a  series  of  tubes  of  fluid  media,  one 
kind  of  bacteria  outgrowing  the  rest.  Isolation  was  also  ac- 
complished by  diluting  the  material  very  highly  and  then  in- 
oculating one  drop  into  each  of  a  large  number  of  tubes  of 
broth.  Some  tubes  would  thus  receive  no  bacteria,  others  would 
receive  several,  and  occasionally  one  would  receive  only  a  single 
germ  and  would  give  rise  to  a  pure  culture.  Another  early 
method  of  separating  a  pathogenic  species  was  by  inoculation 
of  animals.  The  ability  of  the  animal  to  prevent  the  development 
of  all  but  one  species  contained  in  the  inoculated  material  was 
utilized  to  obtain  the  first  pure  cultures  of  anthrax  bacilli  and 
tubercle  bacilli.  These  methods  are  successfully  employed  only 
for  relatively  few  bacterial  species. 


IO6  BACTERIOLOGY 

Methods  of  isolating  bacteria,  which  are  of  more  general  appli- 
cation, were  introduced  by  Koch.  The  essential  characteristic 
of  these  methods  is  the  dilution  of  the  bacteria  in  a  fluid  medium 
which  quickly  becomes  solid  so  that  each  germ  develops  in  a 
definite  fixed  position  in  the  medium.  The  great  progress  which 
bacteriology  has  made  during  the  last  twenty  years  is  largely 
owing  to  these  methods. 

It  is  impossible  in  most  cases  to  distinguish  between  bacteria 
of  different  varieties  by  microscopical  examination  alone.  Bac- 
teria of  widely  different  species  and  quite  unlike  one  another  in 
their  properties  may  present  similar  appearances  under  the  mi- 
croscope. The  differences  which  they  exhibit  are  usually  appar- 
ent when  they  are  grown  in  culture-media.  The  growth,  called 
a  colony,  which  results  from  the  multiplication  of  a  single 
bacterium,  is  in  many  cases  very  characteristic  for  the  species. 
By  the  plate-method,  the  individual  bacteria  in  a  mixture  are 
separated  from  one  another  by  dilution.  They  are  fixed  in  place 
by  the  use  of  a  solid  medium.  They  are  allowed  to  grow,  and 
from  each  individual  there  arises  a  colony.  It  is  usually  possible 
to  distinguish  between  colonies  arising  from  different  species  when 
it  is  not  possible  to  distinguish  between  the  individual  bacteria 
of  these  species.  A  convenient  comparison  has  been  suggested  by 
Abbott.  A  number  of  seeds  of  different  sorts  may  appear  very 
much  alike,  and  considerable  difficulty  may  be  found  in  distin- 
guishing one  from  another  with  the  eye.  Let  them  be  sown,  how- 
ever, and  let  plants  develop  from  them,  and  these  plants  will 
easily  be  distinguished  from  one  another.1 

Method  of  Making  Plate -cultures. — Melt  three  tubes  of  gela- 
tin or  agar.  (There  is  some  difficulty  in  keeping  agar  in  a  fluid 
state  while  dilutions  are  being  made.  It  is  necessary  to  have 
some  form  of  water-bath  with  a  thermometer  for  the  purpose.) 
Let  the  liquefied  agar  cool  to  45°  C.  Gelatin  may  be  used  at  a 

1  It  must  be  understood  that  no  close  comparison  can  be  drawn  between  higher 

"ally  present  in  the 
the  progeny  of  one 


'rb     l/U-70-     l/C>     U'/  U/UUrb     U&I/W&&HP     ilrvgiwi 

plants,  which  simply  complete  the  development  of  parts  potentially  present  in  the 
seed,  and  colonies  of  bacteria,  which  are  aggregates  of  individuals, 


individual  of  the  same  kind. 


THE    CULTIVATION    OF   MICRO-ORGANISMS  IOy 

temperature  anywhere  between  28°  and  40°  C.  Take  a  small 
portion  of  the  material  to  be  examined — pus,  for  example — and- 
introduce  it  with  a  sterilized  platinum  wire  or  loop  into  one  of 
the  tubes.  The  plug  of  the  test-tube  is  to  be  withdrawn,  twisting 
it  slightly,  taking  it  between  the  third  and  fourth  fingers  of  the 
left  hand,  with  the  part  that  projects  into  the  tube  pointing  to- 
ward the  back  of  the  hand.  It  must  not  be  allowed  to  touch 
any  object  while  the  inoculation  is  going  on.  Pass  the  neck  of 
the  tube  through  the  flame.  If  any  of  the  cotton  adheres  to  the 
neck  of  the  tube,  pull  the  cotton  away  with  sterilized  forceps, 
while  the  neck  of  the  tube  touches  the  flame,  so  that  the  threads 
of  cotton  may  be  burned  and  not  fly  into  the  air  of  the  room. 


FIG.  39. — Method  of  inoculating  culture  media. 

The  tube  is  held  as  nearly  horizontal  as  possible.  The  tube  is 
to  be  held  in  the  left  hand  between  the  thumb  and  forefinger, 
the  tube  resting  upon  the  palm,  and  the  neck  of  the  tube  pointing 
upward  and  to  the  right.  Mix  the  material  introduced  thor- 
oughly with  the  liquefied  culture-medium,  taking  care  not  to  wet 
the  plug.  Now  remove  the  plug  again,  and,  having  sterilized  the 
platinum  wire,  insert  it  into  the  liquefied  medium.  Carry  three 
loopfuls  in  sucession  from  thistube/whichisNo.  i,  into  tube  No.  2. 
When  two  tubes  are  being  used  at  the  same  time,  they  are 
placed  side  by  side  between  the  thumb  and  forefinger  of  the  left 
hand.  The  two  plugs  are  held  between  the  second  and  third  and 
the  third  and  fourth  fingers  of  the  left  hand,  respectively.  The 
wire  may  now  be  passed  into  the  first  tube,  which  we  will  suppose 


io8 


BACTERIOLOGY 


to  hold  some  material  containing  bacteria,  and  a  little  of  this 
material  may  be  removed  on  the  tip  of  the  wire  from  the  first 
tube  to  the  second.  When  the  needle  is  introduced  into  or  re- 
moved from  either  tube  it  should  not  touch  the  side  of  the  tube 
at  any  point,  and  should  only  come  in  contact  with  the  region 
desired.  After  inoculation  of  the  second  tube  has  been  effected, 
the  wire  is  to  be  heated  to  a  red  heat  in  the  flame,  the  necks  of 
the  tubes  are  to  be  passed  through  the  flame,  and  the  plugs  are  to 
be  returned  to  their  respective  tubes.  In  the  same  manner 
transfer  three  loopfuls  from  tube  No.  2  into  tube  No.  3.  The 
original  material  will  obviously  be  diluted  in  tube  No.  i,  more  in 
tube  No.  2,  and  still  more  in  tube  No.  3.  The  most  convenient 
form  of  plate  is  that  known  as  a  Petri  dish,  a  small  glass  dish 


FIG.  40. — Petri  dish. 

about  10  cm.  in  diameter  and  1.5  cm.  in  height,  provided  with  a 
cover  which  is  a  little  larger  but  of  the  same  form.  This  dish 
should  be  cleaned  and  sterilized  for  an  hour  in  a  hot-air  sterilizer 
at  1 50°  C.  or  higher.  When  it  is  cool  it  may  be  used. 

Such  dishes  having  previously  been  prepared,  the  contents  of 
tube  No.  i  are  poured  into  one  dish,  and  those  of  tube  No.  2 
into  another,  and  those  of  tube  No.  3  into  a  third.  They  are  to 
be  labeled  Nos.  i,  2,  and  3.1  In  pouring  proceed  as  follows: 
remove  the  plug  of  tube  No.  i ;  heat  the  neck  of  the  tube  in  the 
flame;  allow  it  to  cool,  holding  it  in  a  nearly  horizontal  position. 
When  the  tube  has  cooled,  lift  the  cover  of  the  Petri  dish  a  little, 
holding  it  over  the  dish;  pour  the  contents  of  tube  No.  i  into  the 
dish,  and  replace  the  cover  of  the  dish.  The  interior  of  the  dish 

1  The  labels  should  be  moistened  with  the  finger,  which  has  been  dipped  in  water. 
They  should  not  be  licked  with  the  tongue.  While  working  in  the  bacteriological 
laboratory  it  is  best  to  make  it  a  rule  that  no  object  is  to  be  put  in  the  mouth. 


THE    CULTIVATION    OF    MICRO-ORGANISMS  1 09 

should  be  exposed  as  little  and  as  short  a  time  as  possible.  Tubes 
Nos.  2  and  3  are  to  be  treated  in  the  same  manner.  Burn  the 
plugs,  and  immerse  the  empty  tubes  in  5  per  cent  solution  of 
carbolic  acid.  Where  much  culture  work  is  being  done,  it  will  be 
found  convenient  to  sterilize  the  mouth  of  each  tube  by  thorough 
heating  in  the  flame  after  pouring  out  its  contents,  and  then  to 
replace  the  plug.  The  tube  may  then  be  placed  in  a  special 
receptacle  which  is  sterilized  with  its  contents  in  the  autoclave 
at  120°  C.  for  20  minutes,  at  the  end  of  the  day's  work. 


FIG.  41. — Colonies  in  gelatine  plate  showing  how  they  may  be  separated  and  the 

organisms  isolated. 

The  culture-medium  in  the  Petri  dish  will  soon  solidify. 
Petri  dishes  of  agar  should  be  inverted  after  the  medium  is  firmly 
set;  otherwise  the  water,  which  evaporates  from  the  surface  and 
condenses  on  the  inside  of  the  lid,  may  overflow  the  surface  of 
the  agar,  confusing  the  result.  Agar  plates  are  usually  developed 
in  the  incubator.  Gelatin  plates  must  be  developed  at  a  tempera- 
ture below  the  melting-point  of  the  medium,  which  is  usually 
between  22°  and  28°  C.  Colonies  usually  appear  in  from  one  to 
two  days.  In  plate  No.  i  they  will  be  very  numerous,  in  plate 


no 


BACTERIOLOGY 


No.  2  less  numerous,  and  in  plate  No.  3  still  less  numerous. 
Where  the  number  is  small  the  colonies  will  be  widely  separated 
and  can  readily  be  studied.  They  may  be  examined  with  a  hand- 
lens,  or  the  entire  dish  may  be  placed  on  the  stage  of  the  micro- 
scope and  the  colonies  be  inspected  with  the  low  power.  The 
iris  diaphragm  should  be  nearly  closed  and  the  plane  mirror 
should  be  used.  Dilution-cultures  prepared  as  described  in  the 
next  paragraph,  where  the  principle  is  the  same,  are  shown  in 
Fig.  43.  In  tube  No.  i  the  colonies  are  so  numerous  as  to  look 
like  fine  white  dust.  In  tubes  2  and  3  they  become  less  numerous 
and  larger. 

Esmarch's  Roll-tubes. — Use  liquefied  gelatin  or  agar.     The 
dilutions  in  tubes  i,  2  and  3  are  made  as  above.      Tubes  contain- 


FIG.  42. — Manner  of  making  Esmarch  roll- tube. 

ing  a  rather  small  amount  of  the  culture-medium  are  more  con- 
venient. A  block  of  ice  should  be  at  hand,  and,  with  a  tube  filled 
with  hot  water  and  lying  horizontally,  a  hollow  of  the  size  of  the 
test-tube  should  be  melted  on  the  upper  surface  of  the  ice.  In 
this  hollow,  place  the  tube  of  liquefied  gelatin  or  agar;  roll  it  rapid- 
ly with  the  hand,  taking  care  that  the  culture-medium  does  not 
run  toward  the  neck  as  far  as  the  cotton  plug.  The  medium  is 
spread  in  a  uniform  manner  around  the  inside  of  the  tube,  where 


THE    CULTIVATION    OF   MICRO-ORGANISMS 


III 


it  becomes  solidified.     Gelatin  roll-tubes  must  be  kept  in  a  place 
so  cool  that  there  is  no  danger  of  their  melting;  in  handling  them 


FIG.  43. — Dilution-cultures  in  Esmarch  roll-tubes.  In  tube  i  the  colonies  are 
very  close  together;  in  tube  2  they  are  somewhat  separate;  in  tube  3  they  are  well 
isolated. 

they  are  to  be  held  near  the  neck,  so  that  the  warmth  of  the  hand 
may  not  melt  the  gelatin.     Agar  roll- tubes  should  be  kept  in  a 


112  BACTERIOLOGY 

position  a  little  inclined  from  the  horizontal,  with  the  neck  up, 
for  twenty-four  hours,  so  that  the  agar  may  adhere  to  the  wall 
of  the  tube. 

By  the  plate-method  as  originally  devised  by  Koch,  instead  of  using 
Petri  dishes,  the  gelatin  was  poured  upon  a  sterile  plate  of  glass.  This  plate 
of  glass  was  laid  on  another  larger  plate  of  glass,  which  formed  a  cover  for  a 
dish  of  ice-water,  the  whole  being  provided  with  a  leveling  apparatus.  The 
plate  was  kept  perfectly  level  until  it  had  solidified,  which  took  place  rapidly 
on  the  cold  surface.  The  glass  plates  were  placed  on  little  benches  enclosed 
within  a  sterile  chamber.  The  more  convenient  Petri  dish  has  now  displaced 
the  original  glass  plate. 

Streak  Method  of  Isolating  Bacteria. — The  isolation  of  bac- 
teria may  sometimes  be  effected  by  drawing  a  platinum  wire 
containing  material  to  be  examined  rapidly  over  the  surface  of  a 
Petri  dish  containing  solid  gelatin  or  agar;  or  over  the  surface  of 
the  slanted  culture-medium  in  a  test-tube;  or  by  drawing  it  over 
the  surface  of  the  medium  in  one  test-tube,  then,  without  steril- 
izing, over  the  surface  of  another,  perhaps  over  several  in  succession. 
This  method  is  ordinarily  less  reliable  than  the  regular  plating 
method. 

Veillon's  Tall-tube  Method. — Three  to  six  tubes  of  glucose 
agar,  the  agar  being  at  least  6  cm.  deep,  are  liquefied  and  cooled 
to  45°  C.  in  a  water-bath.  A  small  amount  of  the  material  to 
be  examined  is  placed  in  the  first  tube  by  means  of  the  platinum 
loop,  and  carefully  mixed.  From  this  dilutions  are  made  in  series 
to  tubes,  2,  3,  4,  5  and  6,  each  being  carefully  mixed  without  intro- 
ducing air  bubbles.  The  tubes  are  quickly  solidified  by  immersion 
in  cold  water,  and  are  incubated  at  37°  C.  These  culture  tubes 
offer  the  contained  bacteria  a  wide  range  of  oxygen  supply. 
This  is  abundant  at  and  near  the  top,  and  gradually  diminishes 
lower  in  the  tube  until  near  the  bottom  almost  perfect  anaerobic 
conditions  obtain.  The  method  is  very  useful  in  isolating  B. 
bifidus  from  feces  of  infants,  and  in  studying  the  oxygen  require- 
ments of  other  bacteria.  When  energetic  gas-forming  bacteria 
are  present  in  considerable  number,  the  method  is  of  no  value. 


THE    CULTIVATION   OF   MICRO-ORGANISMS  113 

Colonies  are  picked  out  with  sterile  glass  capillaries,  and  deeper 
colonies  are  reached  by  breaking  the  tube.  The  successful  use 
of  the  method  requires  some  practice. 

Appearance  of  the  Colonies. — The  colonies  obtained  in  the 
Petri  dishes  or  roll-tubes  (Fig.  43)  may  be  studied  with  a  hand- 
lens  or  with  a  low  power  microscope.  In  the  latter  case,  use  the 
plane  mirror  with  the  iris  diaphragm  nearly  closed.  The  colonies 
present  various  appearances.  Some  of  them  are  white,  some 
colored;  some  are  quite  transparent  and  others  are  opaque;  some 
are  round,  some  are  irregular  in  outline;  some  have  a  smooth 
surface,  others  appear  granular,  and  others  present  a  radial 
striation.  Surface  colonies  often  present  different  appearances 
from  those  occurring  more  deeply.  Surface  colonies  are  likely 
to  be  broad,  flat  and  spreading.  If  the  colony  consists  of  bacteria 
which  have  the  property  of  liquefying  gelatin,  a  little  funnel- 
shaped  pit  or  depression  forms  at  the  site  of  the  colony.  The 
appearance  of  colonies  may  be  of  great  assistance  in  determining 
the  character  of  doubtful  species.  The  appearance  in  gelatin 
plates  of  the  colonies  of  the  spirillum  of  Asiatic  cholera,  for  in- 
stance, is  one  of  the  most  characteristic  manifestations  of  this 
organism. 

Pure  Cultures. — From  these  colonies  pure  cultures  may  be 
obtained  by  the  process  called  "fishing."  Select  a  colony  from 
which  cultures  are  to  be  made;  touch  it  lightly  with  the  tip  of  a 
sterilized  platinum  wire,  taking  great  care  not  to  touch  the  me- 
dium at  any  other  point.  Introduce  the  wire  into  a  tube  of  gelatin 
after  removing  the  plug  and  flaming  the  mouth  of  the  tube. 
Sterilize  the  wire  and  plug  the  tube.  In  a  similar  manner,  and 
from  the  same  colony,  inoculate  tubes  of  agar,  bouillon,  milk, 
potato  and  blood-serum.  Gelatin  tube  cultures  are  usually  inocula- 
ted by  introducing  the  platinum  needle  into  the  medium  vertically, 
making  a  "stab-culture."  Inclined  surfaces  such  as  those  of 
agar,  potato  or  blood-serum  are  inoculated  by  drawing  the  wire 
lightly  over  the  surface  of  the  medium,  making  a  "smear-culture" 
or  "streak-culture"  (Figs.  44  and  45).  Liquid  media  are  inocula- 
8 


BACTERIOLOGY 

ted  by  simple  introduction  of  a  small  mass  of  bacteria  and  mixing 
them  with  the  medium.  At  the  same  time  it  is  well  to  make  a 
smear  preparation  from  the  colony  and  to  stain  with  one  of  the 
aniline  dyes  so  as  to  determine  the  morphology  of  the  bacteria. 
The  growths  which  take  place  in  the  tubes  should  contain  one 
and  the  same  kind  of  bacteria.  As  seen  under  the  microscope 
these  bacteria  should  have  the  same  general  form  and  appearance 


FIG.  44. — Stab-culture. 

A  rubber  stopper  may 
be  used  to  prevent  drying, 
see  page  121. 


FIG.  45. — Smear-culture. 
This    tube    shows    the 
rubber  cap  used  to  prevent 
drying. 


as  those  seen  in  the  colony  from  which  they  were  derived.  This 
will  be  the  case,  provided  the  colony  has  resulted  from  the  develop- 
ment of  a  single  bacterium. 

A  pure  culture  is  a  culture  which  contains  only  the  descendants 
of  a  single  cell. 

Stock  Cultures. — To  maintain  their  vitality  bacteria  need  to 
be  transplanted  from  one  tube  to  another  occasionally;  the  time 


THE   CULTIVATION   OF   MICRO-ORGANISMS  11$ 

varies  greatly  with  different  species.  Many  bacteria  grow  on 
culture-media  with  difficulty  at  the  first  inoculation,  but  having^ 
become  accustomed  to  their  artificial  surroundings,  as  it  were, 
they  may  be  propagated  easily  afterward;  this  is  especially  true 
of  the  tubercle  bacillus.  After  they  are  developed,  stock  cultures 
are  best  kept  in  a  refrigerator,  and  it  is  well  to  seal  them  so  as  to 
prevent  drying.  Rubber  caps  or  rubber  stoppers  are  useful  for 
this  purpose  (Figs.  44  and  45). 

Some  bacteria  flourish  better  on  one  culture-medium  than 
another.  The  tubercle  bacillus  grows  best  on  blood-serum  and 
glycerin-agar;  the  bacillus  of  diphtheria  grows  best  on  Loffler's 
blood-serum;  the  gonococcus  on  human  serum-agar  or  ascitic- 
fluid-agar. 

The  virulence  of  most  pathogenic  bacteria  becomes  diminished 
after  prolonged  cultivation  upon  media.  In  some  forms  the  viru- 
lence is  lost  very  quickly,  for  example,  the  Streptococcus  pyo genes 
and  Micrococcus  lanceolatus  of  pneumonia. 

REGULATION  or  TEMPERATURE. 

High-temperature  Incubator. — Many  bacteria  flourish  best 
at  a  temperature  about  that  of  the  human  body,  37°  C.  Some 
species  will  grow  only  at  this  temperature.  The  pathogenic  bac- 
teria in  particular,  for  the  most  part,  thrive  best  at  a  point  near 
the  body  temperature. 

The  ordinary  incubator  is  a  box  made  of  copper,  having 
double  walls,  the  space  between  the  two  being  filled  with  water. 
The  outer  surface  is  covered  with  some  non-conductor  of  heat, 
such  as  felt  or  asbestos.  At  one  side  is  a  door,  which  is  also  double. 
The  inner  door  is  of  glass,  the  outer  door  is  of  copper  covered 
with  asbestos.  At  one  side  is  a  gauge  which  indicates  the  level 
at  which  the  water  stands  in  the  water-jacket.  The  roof  is  per- 
forated with  several  holes,  some  of  which  permit  the  circulation 
of  the  air  in  the  air-chamber  inside  the  box;  some  of  them  enter 
the  water-jacket.  A  thermometer  passes  through  one  of  these 


n6 


BACTERIOLOGY 


holes  into  the  interior  of  the  air-chamber,  and  often  another  into 
the  water  standing  in  the  water-jacket.  A  gas-regulator  passes 
through  another  hole,  and  is  immersed  in  the  water  standing  in 
the  water-jacket.  There  are  various  forms  of  gas-regulators 


FIG.  46. — Incubator. 

more  or  less  complicated.  The  simplest  and  least  expensive 
thermo-regulators  for  gas  are  made  of  glass  and  filled  with  mercury 
or  with  mercury  and  some  lighter  liquid,  the  heavy  mercury 
serving  to  close  the  chief  source  of  gas  supply  when  the  desired 


THE   CULTIVATION   OF   MICRO-ORGANISMS 


temperature  has  been  attained,  while  a  minute  opening  at  another 
point  remains  open  to  furnish  sufficient  gas  to  keep  the  flame 
alight,  but  not  sufficient  to  maintain  the  temperature.  Upon 
cooling  the  mercury  falls  and  allows  gas  to  flow  again  through 
the  larger  opening.  In  this  way  the  supply  of  gas  is  made  large 
whenever  the  temperature  is  a  little  below  the 
desired  temperature  and  very  small  whenever 
the  temperature  rises  above  that  point,  and  the 
temperature  varies  within  a  slight  range.  The 
Reichert  regulator  is  designed  to  operate  ac- 
cording to  these  principles,  and  various  modi- 
fications of  this  regulator  are  on  the  market. 
In  many  of  these  instruments  the  larger  supply 
is  only  imperfectly  shut  off  at  the  desired  tem- 
perature, and,  where  the  weight  of  the  mercury 
is  relied  upon  to  stop  this  opening,  the  gas  may 
often  bubble  out  through  it  unless  special  pre- 
cautions are  taken  to  regulate  the  pressure  of 
the  gas  supply. 

A  modification  of  this  type  of  regulator 
devised  by  Mac  Neal1  overcomes  this  difficulty 
(see.  Fig.  48).  The  inlet  tube  A  leads  through 
the  wall  of  the  chamber  D,  to  which  it  is  fused, 
into  an  inner  upright  tube,  B  C .  Near  the  upper 
end  of  this  upright  is  a  small  opening,  O,  which  allows  the  minimum 
supply  of  gas  to  pass  to  the  burner  to  avoid  extinction  of  the  flame. 
The  lower  end  of  this  upright  tube  fits  quite  closely  the  bottom  of 
the  chamber  D,  around  the  opening  leading  into  the  capillary 
tube,  EF.  This  end  is  adjusted  so  close  to  the  bottom  that  mer- 
cury will  not  pass  through  between  inner  and  outer  tube  at  less 
than  twenty  millimeters  mercury  pressure,  yet  not  so  close  but 
that  an  abundant  supply  of  gas  may  pass.  The  proper  adjust- 
ment of  this  part  must  be  thoroughly  tested  before  the  instrument 
leaves  the  factory.  The  upper  end  of  the  upright,  BC,  is  closed 

1  The  Anatomical  Record,  August,  1908,  Vol.  II,  No.  5. 


FIG.  47. — Reichert's 
gas-regulatcr. 


n8 


BACTERIOLOGY 


by  a  ground  glass  stopper,  which  also  closes  the  top  of  the  outer 
chamber,  D.  In  the  ground  surface  of  this  stopper  a  gamma- 
shaped  (r)  groove  is  cut,  the  vertical  limb  extending  from  the 
lower  tip  of  the  stopper  to  the  level  of  the  opening,  O.  The 
horizontal  limb  is  deep  where  it  joins  the  ver- 
tical, but  gradually  becomes  shallow  and  ends 
about  one-quarter  the  way  around  the  stopper. 
This  groove  serves  for  passage  of  the  gas  from 
the  inner  tube  BC,  to  the  opening  O,  and  thus 
to  the  outer  chamber  D,  and  by  rotating  the 
stopper,  the  amount  of  gas  flowing  through 
this  passage  may  be  reduced  to  any  desired 
point.  The  outlet  tube,  H,  leads  from  the 
chamber  D  to  the  burner  connection. 

The  capillary,  EF,  leads  to  a  bulb  of  suffi- 
cient size;  the  larger  the  more  sensitive  the 
instrument.  Either  the  large  bulb  with  inside 
capillary,  J,  to  be  filled  with  mercury  and 
alcohol,  or  the  smaller  simple  bulb  for  mercury 
alone  may  be  used.  A  side  arm  is  attached 
to  one  side  of  the  capillary  EF,  for  conveni- 
ently controlling  the  height  of  the  mercury 
column.  Either  the  curved  capillary  tube 
with  stopcock  and  a  cup  on  the  end,  or  the 
simple  tube  with  metal  screw  cemented  in, 
may  be  used  here,  according  to  the  purpose 
which  the  regulator  is  to  serve.  These  parts 
FIG.  48.— Mac  Neal  are  simiiar  to  those  of  Novy's  modification  of 

gas-regulator. 

the  Reichert  regulator. 

To  fill  the  instrument,  the  air  is  partly  driven  out  by  heating 
the  bulb  and  then  the  desired  liquid  is  drawn  in  by  cooling, 
repeating  the  heating  and  cooling  until  the  instrument  is  full 
of  the  liquid.  For  the  small  bulb,  mercury  is  always  used  alone. 
The  large  bulb,  on  the  other  hand,  is  filled  first  with  either  ether, 
alcohol  or  toluol,  and  then  part  of  this  liquid  is  forced  out  by 


THE   CULTIVATION   OF   MICRO-ORGANISMS 


IIQ 


heat  and  replaced  with  mercury  so  that  the  capillary  EF,  the  bulb 
at  its  lower  end,  and  a  small  part  of  the  large  bulb  J,  are  occupied, 
by  the  mercury.     Ether  may  be  used  when  the  regulator  is  not 
to  be  heated  above  35°  C.,  alcohol  when  it  is  not  to  be  heated 
above  75°  C.,  and  toluol  for  temperatures  between  75°  and  ico°  C. 
A  more  satisfactory  regulator  is  that  of  Roux.     It  is  con- 
structed entirely  of  metal,  and  its 
operation  is   due   to  the  unequal 
expansion  and  contraction  of  two 
metals  which  are  riveted  together. 
Fig.  49  shows  this  regulator.     The 
gas  passes  in  at  e  and  passes  out 
at  d.     The  amount  of  gas  passing 
through  is  regulated  by  a  piston 
on  the  end  of  the  set  screw  inside 


FIG.  49. — Roux  bime- 
tallic gas-regulator,  a,  Set 
screw;  b,  Screw  collar;  c, 
Clamp;  d,  Outlet  for  gas; 
e,  Inlet  for  gas. 


FIG.  50. — Koch  auto- 
matic gas-burner. 


the  tube  from  which  the  outlet  tube  branches  off.  This  piston 
moves  in  or  out  according  to  the  changes  of  temperature  of  the 
water  jacket  of  the  incubator  into  which  the  stem  (/")  of  the  regu- 
lator is  inserted.  This  stem  is  fenestrated  and  has  the  riveted 
metallic  strips  running  down  in  it.  These  strips  are  pivoted  at 
the  collar,  g. 


1 20  BACTERIOLOGY 

The  gas  coming  from  the  gas-regulator  passes  to  a  Bunsen 
burner,  which  stands  underneath  the  incubator.  This  burner 
should  have  some  kind  of  automatic  device  for  cutting  off  the  flow 
of  gas  in  case  it  becomes  accidentally  extinguished  by  a  sudden 
draught  of  air  or  from  any  other  cause.  The  automatic  burner 
invented  by  Koch  is  an  ingenious,  simple  and  effective  device 
(Fig.  50).  The  coils  of  metal -seen  on  each  side  at  the  top  of 
the  burner  are  so  arranged  that  when  they  expand  they  turn  the 
disk  below  so  as  to  support  the  arm  coming  from  the  stop-cock; 
when  they  cool  they  turn  the  disk  in  the  opposite  direction,  and 
allow  the  arm  to  fall  and  cut  off  the  gas.  Some  inconvenience 
will  at  times  arise  from  irregularities  in  the  flow  of  gas  from  the 
main  supply-pipe.  A  properly  constructed  regulator  should, 
however,  compensate  perfectly  for  all  ordinary  variations  in  pres- 
sure of  artificial  gas.  Natural  gas  is  commonly  furnished  at 
much  higher  pressure  and  it  is  necessary  to  install  apparatus 
to  reduce  the  pressure,  a  gas-pressure  regulator,  between  the  gas 
main  and  the  thermoregulator.  Fluctuations  of  the  temperature 
within  the  incubator  depend  very  largely  upon  the  external 
temperature,  especially  if  its  outer  walls  are  not  well  insulated. 
The  incubator  should,  therefore,  be  kept  in  a  place  free  from 
draughts  of  air,  where  the  temperature  is  fairly  constant. 

In  large  modern  laboratories,  the  incubators  are  built  in  as 
special  insulated  rooms,  heated  by  a  gas  stove.  A  regulator  of 
large  size  is  installed  to  control  the  supply  of  gas  to  the  stove. 
These  incubator  rooms  are  very  satisfactory  and  provide  quite  a 
range  of  constant  temperature  according  to  the  height  of  shelves 
from  the  floor. 

Culture-tubes  which  are  being  kept  in  the  incubator  are  likely 
to  become  dry  if  their  stay  is  prolonged.  In  such  cases  they 
should  be  covered  with  rubber  caps,  tin-foil,  sealing-wax,  paraffin, 
or  some  other  device  to  prevent  evaporation.  If  rubber  caps 
are  used,  they  should  be  left  in  i-iooo  bichloride  of  mercury 
solution  for  an  hour,  and  the  cotton  plugs  should  be  singed  in  the 
flame,  before  putting  them  on  (Fig.  45).  Some  bacteriologists 


THE   CULTIVATION    OF   MICRO-ORGANISMS  121 

prefer  rubber  stoppers,  which  may  be  boiled  and  stored  in  bi- 
chloride of  mercury  solution.     Cut  the  cotton  plug  even  with  the- 
edge  of  the  tube;  singe  it  in  the  flame;  push  it  into  the  tube  about 
i  cm.,  and  insert  the  rubber  stopper     (Fig.  44). 

Low-temperature  Incubator. — An  incubator  regulated  for  so- 
called  "room  temperature"  is  very  desirable  for  the  cultivation 
of  bacteria  upon  gelatin  and  for  the  bacteriological  analysis 
of  water.  In  our  climate  the  temperature  of  the  rooms  of  the 
laboratory  often  reaches  a  point  at  which  gelatin  melts,  and 
for  this  reason  in  a  low-temperature  incubator  provision  has  to 
be  made  for  cooling  when  the  room  temperature  is  too  high  as 
well  as  for  heating  when  it  is  too  low. 

A  form  of  incubator  devised  by  Rogers1  for  this  purpose 
consists  of  a  refrigerator  or  of  a  specially  constructed  chamber 
heated  by  electricity  and  controlled  by  an  electric  thermoregu- 
lator.  Below  is  given  a  description  of  an  incubator  constructed 
according  to  Rogers'  plans.  This  incubator  has  been  in  use 
for  some  time  and  has  given  entire  satisfaction  since  the  pre- 
cautions noted  below  were  followed.  There  would  appear  no 
reason  why  this  incubator  should  not  be  employed  for  high 
temperatures  as  well  as  for  low,  but  so  far  it  has  been  run  at  22°  C. 
The  temperature  has  kept  very  constant.  The  incubator  con- 
sists of  a  refrigerator,  30  inches  high,  24  inches  wide,  18  inches 
from  front  to  back,  all  outside  measurements.  Instead  of  the 
ordinary  drip  pipe,  there  is  a  coil  of  i-inch  galvanized  iron  pipe 
run  down  the  back  of  the  cooling  chamber  attached  water-tight 
to  the  ice  tank.  From  the  bottom  of  the  cooling  chamber  the 
coil  runs  up  perpendicularly  nearly  to  the  bottom  of  the  ice 
compartment,  and  then  runs  horizontally  through  the  wall  of  the 
refrigerator.  A  bracket  on  the  outside  supports  a  drip-pan. 
A  thermometer  encased  in  a  fenestrated  metal  jacket  is  inserted 
about  half  way  up  on  one  side.  A  lump  of  ice,  about  50  pounds, 
placed  in  the  ice  compartment  serves  to  keep  the  tem- 

1 L.  A.  Rogers.  On  electrically  controlled  low  temperature  incubators.  Cen- 
tralblatt  fur  Bakteriologie,  etc.,  Bd.  XV,  Abt.  II,  pp.  236-239,  Sept.  23,  1905. 


122 


BACTERIOLOGY 


perature  sufficiently  cool.  In  summer  doubtless  more  ice  will  be 
required. 

For  heating,  an  ordinary  i6-candle-power  electric  bulb  is 
used,  and  the  electricity  is  obtained  from  the  public  supply. 
The  wire  is  run  through  one  of  the  walls,  and  a  part  of  the  current 
is  made  to  operate  a  horse-shoe  magnet,  and  another  part  is 
conducted  through  the  lamp  used  for  heating. 

The  accompanying  diagram  (Fig.  51),  will  serve  to-  show 
the  arrangement. 

A  telegraph  key  is  used  to  supply  the  horse-shoe  magnet 


Ther.morea/ulator 
FIG.  51. — Diagram  of  electric  regulator  for  low- temperature  incubator. 

which  is  inserted  in  the  heating  circuit  in  such  a  way  that  when 
the  armature  is  attracted  toward  the  magnet  the  circuit  is  com- 
pleted and  the  lamp  is  consequently  lighted.  The  part  of  the 
current,  a,  supplying  the  magnet  first  passes  through  a  small 
lamp  and  through  two  resistance  coils  so  as  to  red  ace  the  current. 
It  then  passes  through  the  magnet,  and  is  continued  on  to  the 
set-screw,  b,  which  is  so  placed  that  when  the  thermoregulator 
comes  in  contact  with  it  the  circuit  is  complete.  The,  regu- 
lator consists  of  a  strip  of  hard  rubber  and  a  strip  of  brass  riveted 


THE   CULTIVATION   OF   MICRO-ORGANISMS  123 

together.  One  end  is  fixed,  while  the  other  is  free,  and  when  it 
is  heated  it  tends  to  bend  toward  the  metal  side,  when  it  cools  it 
bends  toward  the  rubber.  The  brass  strip  is  15  inches  long,  J 
inch  thick,  and  \  inch  wide;  the  rubber  strip  is  \  inch  thick,  \ 
inch  wide,  and  a  little  less  than  15  inches  long.  In  the  diagram 
the  end  is  fixed  at  d  and  is  free  at  b.  When  it  is  heated,  the  free 
end  travels  away  from  the  set-screw  at  6;  when  it  cools,  it  moves 
toward  the  set-screw.  Rogers  also  recommends  a  regulator 
made  of  invar  and  brass  instead  of  hard  rubber  and  brass.  Where 
invar  is  used  instead  of  the  hard  rubber  the  dimensions  for  the 
two  metals  are  the  same  as  those  given  for  the  brass  strip  in  the 
hard-rubber-brass  regulator  just  described.  As  is  evident  from 
the  description,  the  circuit  controlling  the  magnet  is  closed  when- 
ever the  free  end  of  the  regulator  comes  in  contact  with  the  set 
screw  at  b.  When  this  circuit  is  closed  the  magnet  attracts  the 
armature,  and  the  heating  circuit  is  closed  by  the  contact  formed 
at  c  between  the  armature  and  the  set-screw.  In  the  diagram 
this  point  of  contact  is  put  to  one  side  for  the  sake  of  clearness, 
but  as  a  matter  of  fact  in  the  instrument  in  use,  the  set-screw  is 
above  and  between  the  ends  of  the  horse-shoe  magnet,  and 
comes  in  contact  with  the  armature  which  is  extended  upward 
in  the  shape  of  a  tongue.  From  the  description  just  given  it  will 
be  noted  that  the  thermoregulator  does  not  control  the  heating 
directly,  but  indirectly  through  the  electro-magnet. 

Certain  precautions  have  been  found  necessary  in  practice 
in  order  to  obtain  satisfactory  results  with  this  incubator.  The 
set-screw  against  which  the  armature  strikes  at  c  should  be  so 
set  that  the  armature  does  not  come  in  contact  with  the  magnet. 
In  the  apparatus  described  above  there  is  a  space  of  about  J  inch 
between  the  armature  and  the  magnet  when  contact  takes  place 
between  the  set-screw  and  the  armature.  If  the  set-screw  does 
not  project  far  enough  to  prevent  the  armature  from  coming  in 
contact  with  the  magnet,  the  armature  may  adhere  to  the 
magnet  even  after  the  current  is  broken  at  b,  and  when  this  is 
the  case  of  course  the  lamp  remains  lighted,  and  the  temperature 


1 24  BACTERIOLOGY 

may  run  up  too  high.  This  sticking  of  the  armature  to  the  mag- 
net is  said  to  be  due  to  the  residual  magnetism  left  in  the  core  of 
the  magnet.  When  the  current  passing  through  the  magnet  is 
broken  by  the  excursion  of  the  end  of  the  thermoregulator  away 
from  the  set- screw  at  b,  the  armature  is  pulled  away  from  the 
magnet  by  a  coiled  spring.  Another  important  precaution  is 
that  the  points  at  which  contact  is  made  and  broken,  b  and  c, 
should  be  tipped  with  platinum.  A  small  piece  of  platinum 
wire  inserted  into  the  ends  of  the  set-screws  and  a  few  square 
centimeters  of  platinum  foil  riveted  to  the  opposite  point  of  con- 
tact, meet  the  requirements.  If  platinum  is  not  used  at  these 
points  oxidation  takes  place  and  prevents  contact.  The  set- 
screw  at  b  is  set  by  experiment  for  the  temperature  desired. 
The  further  the  point  of  the  set-screw  projects  toward  the  free 
arm  of  the  regulator,  the  higher  the  temperature  maintained. 

CULTIVATION  OF  ANAEROBIC  BACTERIA. 

Deep  Stab  Culture. — Bacteria  which  cannot  grow  in  the  pres- 
ence of  atmospheric  oxygen  may  be  successfully  cultivated  by 
methods  in  which  the  oxygen  is  excluded  or  its  concentration 
diminished.  The  simplest  procedure,  first  practised  by  Liborius, 
is  to  make  deep  stab  cultures  into  freshly  solidified  alkaline 
glucose  agar.  The  agar  quickly  closes  over  the  needle  track  and 
any  traces  of  oxygen  introduced  into  the  depths  of  the  agar  are 
absorbed  and  reduced  by  the  glucose  in  the  presence  of  the 
alkali.  The  bacteria  thus  find  at  various  points  along  the  punc- 
ture all  variations  in  partial  pressure  of  oxygen  from  almost 
complete  absence  up  to  the  concentration  existing  in  the  atmos- 
phere at  the  surface  of  the  medium.  Obligate  anaerobes  begin 
to  grow  near  the  bottom  and,  as  the  gases  produced  replace  the 
air  above,  the  growth  extends  upward,  often  even  entirely  to  the 
surface. 

Veillon  Tube  Cultures. — Isolated  colonies  of  anaerobic  bac- 
teria may  be  obtained  by  a  modification  of  this  tube  method  of 


THE   CULTIVATION   OF   MICRO-ORGANISMS  125 

Liborius,  which  seems  to  have  been  used  first  by  Veillon.  Several 
tubes  of  glucose  agar  are  melted,  cooled  to  45°  C.  and  then  in- 
oculated by  dilution  in  series  just  as  if  plate  cultures  were  to  be 
made.  After  careful  mixing  the  agar  is  quickly  congealed  by 
standing  the  tubes  in  cold  water.  The  later  tubes  in  the  series 
should  contain  only  a  few  bacteria  so  that  single  colonies  may 
develop.  The  method  serves  for  anaerobes  and  also  for  those 
kinds  of  bacteria  which  seem  to  require  some  free  oxygen  but  do 
not  grow  well  when  exposed  to  the  full  amount  in  the  atmosphere 
(B.  abortus,  B.  bifidus). 

Fermentation  Tube. — Anaerobic  bacteria  grow  excellently 
in  the  Smith  fermentation  tube  filled  with  glucose  broth,  especially 
if  a  small  piece  of  naturally  sterile  liver  or  kidney  from  a  small 
animal,  or  a  few  cubic  centimeters  of  naturally  sterile  defibrinated 
blood  be  added  to  the  medium  in  the  tube.  Glucose  gelatin 
to  which  litmus  has  been  added  also  furnishes  a  medium  in  which 
anaerobes  will  grow  abundantly  without  any  special  precautions 
to  protect  them  from  oxygen  or  from  the  air. 

Removal  of  Oxygen. — Anaerobic  conditions  may  be  furnished 
by  pumping  out  the  air  from  a  container  in  which  the  cultures 
have  been  placed,  a  method  employed  by  Pasteur.  The  oxygen 
may  be  absorbed  from  the  air  by  a  mixture  of  pyrogallic  acid 
and  alkali.  Buchner's  method  is  carried  out  as  follows:  Into 
a  bottle  or  tube  which  can  be  tightly  stoppered,  pour  10  c.c.  of  a 
6  per  cent  solution  of  sodium  or  potassium  hydroxide,  for  each 
100  c.c.  of  air  contained  in  the  jar.  Add  one  gram  of  pyrogallic 
acid  for  each  10  c.c.  of  solution.  The  culture-tube  is  placed 
inside  of  the  larger  bottle  or  tube,  supported  above  the  bottom, 
and  the  stopper,  smeared  with  paraffin,  is  inserted.  The  mix- 
ture of  pyrogallic  acid  and  potassium  hydroxide  possesses  the 
property  of  absorbing  oxygen. 

Wright's  Modification  of  Buchner's  method:  The  tube  of  cul- 
ture-medium is  to  be  plugged  with  absorbent  cotton,  using  a  plug 
of  large  size.  The  culture-medium  is  inoculated  in  the  usual 
way.  The  plug  is  cut  off  close  to  the  neck  of  the  tube,  and  is 


126 


BACTERIOLOGY 


then  pushed  into  the  tube  about  i  centimeter.  Now  allow  a 
watery  solution  of  pyrogallic  acid  to  run  into  the  plug,  and  then  a 
watery  solution  of  sodium  or  potassium  hydroxide.  Close 
quickly  and  tightly  with  a  rubber  stopper.  Wright  recommends 
that  the  first  solution  be  freshly  made  and  consist  of  about  equal 

volumes  of  pyrogallic  acid  and  water, 
and  that  the  second  solution  contain  i 
part  of  sodium  hydroxide  and  2  parts 
of  water.  With  6  inch  test-tubes,  f 
inch  diameter,  the  amounts  advised  are 
\  c.c.  solution  of  pyrogallic  acid  and 
i  c.c.  solution  of  sodium  hydroxide. 

Hydrogen  Atmosphere. — The  most 
perfect  anaerobic  conditions  are  ob- 
tained by  replacing  the  air  with  hy- 
drogen in  a  perfectly  air-tight  container. 
The  method  of  hermetically  sealing  such 
containers  full  of  hydrogen  by  melting 
the  glass  in  a  flame  is  really  too  dan- 
gerous to  be  recommended.  The  ap- 
paratus devised  by  Novy  is  most  con- 
venient and  has  practically  superseded 
all  other  devices  for  cultivation  of 
anaerobes  in  hydrogen.  The  Novy  jar 
is  especially  valuable  for  plate  cultures. 
In  using  this  jar,  all  ground-glass  sur- 
faces should  be  thoroughly  coated  with 
a  fairly  stiff  mixture  of  bees  wax  and 
olive  oil  so  as  to  make  all  joints  air-tight.  Rubber  gascots  or 
packing  should  never  be  employed  between  the  ground-glass  sur- 
faces, regardless  of  the  fact  that  many  dealers  furnish  them  for  this 
purpose.  After  the  plate  cultures  or  tubes  have  been  put  into  the 
lower  section  of  the  jar,  the  cover  is  put  on  so  that  the  flanges  fit  to- 
gether perfectly.  A  heavy  rubber  band  may  then  be  passed  around 
the  circumference  of  the  flanges  to  cover  the  circle  of  contact.  Fi- 


FIG.  52. — Arrangement  of 
tubes  for  cultivation  of  anae- 
robes by  Buchner's  method. 


THE    CULTIVATION    OF   MICRO-ORGANISMS 


I27 


pIG    53 — Bottle  for  tube  cultures.     (After  Novy.) 


FIG.  54. — Apparatus  for  Petri  dishes  or  tubes  FIG.  55. — Apparatus  for  plates 

— gasorpyrogallate  method.     (After  Novy.)        or  tubes — gas,  pyrogallate  or  vac- 
uum method.     (After  Novy.) 


128 


BACTERIOLOGY 


nally  two  or  three  clamps,  the  jaws  of  which  are  cushioned  with  cork 
or  with  rubber,  are  fastened  on  the  flanges,  pressing  them  firmly 


FIG.  56. — Tripod  and  siphon  flask  for  anaerobic  culture  by  combined  hydrogen  and 

pyrogallate  method. 

together.  The  jar  is  now  attached  to  a  source  of  pure  hydrogen 
so  that  the  gas  enters  at  the  top  of  the  jar.  The  other  open- 
ing is  connected  with  a  wash  bottle  containing  water  which 


THE    CULTIVATION    OF   MICRO-ORGANISMS  I2Q 

serves  as  a  valve.     Hydrogen  is  passed  through  the  jar  for  two 
hours  or  more.     It  is  well  to  keep  all  flames  away  from  the  appa-_ 
ratus  as  a  precaution  against  explosion  of  the  hydrogen  when 
mixed  with  air. 

The  hydrogen  is  generated  by  the  action  of  25  per  cent 
sulphuric  acid  on  granulated  zinc.  It  should  be  purified  by  pass- 
ing through  a  wash  bottle  of  alkaline  lead  acetate  solution,  a 
second  one  containing  a  solution  of  potassium  permanganate 


FIG.    57. — An  aerobic  organism  (potato  bacillus)  that  will  not  grow  under  a  cover- 
glass. 

and  a  third  of  silver  nitrate.  In  diluting  sulphuric  acid,  the  acid 
must  be  poured  slowly  into  the  water,  and  the  mixture  cooled 
in  a  bath  of  cold  water,  or  under  the  tap.  Carelessness  in  dilut- 
ing this  acid  may  allow  violent  boiling  to  occur,  sometimes  with 
serious  consequences. 

For  critical  work  in  anaerobic  culture  it  is  well  to  combine 
the  pyrogallate  and  hydrogen  methods.  This  is  readily  accom- 
plished by  placing  the  Petri  dishes  on  a  low  glass  tripod  with  a 
small  amount  (2  grams)  of  pyrogallic  acid  beneath  them  on  the 

9 


130  BACTERIOLOGY 

bottom  of  the  Novy  jar.1  On  top  of  the  stack  of  Petrid  is  hesis 
placed  a  small  flask  containing  strong  solution  of  sodium  hydrox- 
ide, and  provided  with  a  siphon  spout  (  see  Fig.  56).  A  rub- 
ber is  attached  to  this  spout  and  leads  down  to  the  floor  of  the 
jar.  After  hydrogen  has  been  passed  through  the  jar  and  it 
has  been  finally  closed,  a 'slight  tipping  to  one  side  starts  the  flow 
of  the  alkali  through  the  siphon  and  so  makes  the  pyrogallic  acid 
available  to  absorb  the  last  traces  of  oxygen. 

Further  Anaerobic  Methods. — Numerous  other  expedients 
have  been  employed  for  the  cultivation  of  anaerobes.  Koch 
covered  part  of  the  surface  of  a  gelatin  plate  with  a  bit  of  steril- 
ized mica  or  a  cover-glass.  Such  a  method  suffices  to  prevent  the 
growth  of  strictly  aerobic  forms  but  rarely  suffices  for  the  success- 
ful culture  of  strict  anaerobes.  Covering  the  surface  of  the  med- 
ium with  sterile  liquid  paraffin  is  a  more  perfect  means  of  exclud- 
ing air. 

In  all  anaerobic  culture  methods,  the  presence  of  one  or  more 
reducing  substances  in  the  culture  medium  is  of  great  importance. 
Those  commonly  employed  are  glucose,  litmus  and  native  protein. 

1MacNeal,  Latzer  and  Kerr,  Journ.  Infect.  Diseases,  1909,  Vol.  VI,  p.  557. 


CHAPTER  VI 
METHODS  OF  ANIMAL  EXPERIMENTATION 

Value  of  Animal  Experimentation. — The  importance  of  ex- 
perimentation upon  animals  in  the  development  of  our  knowledge 
concerning  disease-producing  micro-organisms  can  hardly  be 
over-estimated,  and  animals  must  be  used  in  considerable  numbers 
in  any  adequate  presentation  of  the  subject  to  a  laboratory  class 
in  pathogenic  bacteriology.  Only  in  this  way  has  it  been  possible 
to  discover  the  causal  relation  of  bacteria  to  disease  and  the  way 
in  which  diseases  are  transmitted,  and  it  is  only  by  the  use  of 
animals  that  this  information  can  be  presented  first-hand  to 
students.  The  inoculation  of  animals  also  provides  accurately 
controlled  material  for  studying  the  course  and  termination  of 
the  disease  as  well  as  the  gross  or  microscopic  lesions  produced 
by  it. 

Care  of  Animals. — Laboratory  animals  should  be  housed  in  a 
light,  well-ventilated  room  which  should  be  heated  in  winter  to 
about  60°  F.  If  possible  a  run-way  in  the  open  air  should  be 
provided.  The  fixed  cages  may  be  constructed  with  wood  or 
steel  frames,  but  at  least  the  front  and  preferably  both  front  and 
back  should  be  made  of  strong  wire  netting  to  provide  ample 
ventilation.  For  rats  and  mice  it  is  well  to  provide  an  enclosed 
perfectly  dark  space  inside  the  cage  into  which  these  animals 
may  retire.  Smaller  movable  cages  must  also  be  provided  for 
animals  acutely  sick  and  those  infected  with  dangerously  com- 
municable diseases.  These  must  be  sterilizable,  and  wood  should 
not  be  used  in  their  construction.  Glass  jars  with  weighted 
covers  of  wire  netting  are  useful  for  mice  and  rats,  and  for  larger 
animals  such  as  guinea-pigs,  rabbits  and  cats,  cages  of  galvanized 
iron  and  wire  netting  are  used.  Pigeons  may  also  be  kept  in  such 


132  BACTERIOLOGY 

cages.  Very  large  animals  such  as  monkeys  and  dogs  require 
specially  constructed  cages.  Laboratory  animals  should  receive 
very  careful  attention.  They  should  be  supplied  with  new  food 
at  least  once  daily  and  with  clean  water  twice  a  day.  If  food 
remains  at  the  end  of  the  day,  it  should  be  removed  and  a  smaller 
amount  given  for  the  next  day.  The  cages  should  be  completely 
emptied  and  cleaned  at  least  once  a  week,  the  refuse  being  in- 
cinerated. The  animal  house  should  be  screened,  and  insects  of 
all  kinds  given  careful  attention.  It  will  be  found  practically 
impossible  to  control  the  lice  and  fleas,  but  winged  insects, 
especially  biting  varieties,  may  be  kept  out;  and  bedbugs,  which 
sometimes  gain  entrance  on  new  lots  of  guinea-pigs  or  rats,  should 
not  be  allowed  to  remain  uncontrolled.  These  possible  carriers 
of  infection  require  serious  consideration  as  sources  of  confusion 
where  experimental  investigations  are  being  carried  out,  not  to 
mention  the  element  of  danger  to  the  human  individuals  in  the 
neighborhood. 

Holding  for  Operation. — Animals  to  be  inoculated  or  operated 
upon  must  be  held  in  a  fixed  position.  Many  special  mechanical 
holders  have  been  devised  for  the  various  animals,  but  these 
are  not  necessary  or  especially  useful.  A  pair  of  long-handled 
hemostatic  forceps  with  lock,  or  a  pair  of  placental  forceps  with 
lock,  will  be  found  most  serviceable  in  handling  mice  or  rats, 
the  loose  skin  of  the  animal's  neck  being  caught  in  the  forceps. 
Guinea  pigs  are  best  held  by  an  assistant,  the  thumb  and  fore- 
finger of  one  hand  encircling  the  thorax  just  behind  the  fore  legs 
and  the  other  hand  holding  the  hind  legs  stretched  out.  Rabbits 
are  held  by  the  ears  and  hind  legs  with  the  body  stretched  over 
the  knee.  Monkeys  are  to  be  handled  with  thick  gloves  and 
should  be  caught  around  the  neck  from  behind  with  one  hand  and 
by  the  pelvis  or  hind  legs  with  the  other.  A  second  assistant 
is  required  to  hold  the  fore  legs.  For  all  work  which  would  cause 
any  considerable  pain  the  animal  must  be  anesthetized,  either 
by  putting  it  into  a  closed  compartment  with  the  anesthetic  or 
by  use  of  a  cone.  Anesthesia  is  also  necessary  when  delicate 


METHODS   OF   ANIMAL  EXPERIMENTATION  133 

manipulations  are  to  be  carried  out.  For  operations  requiring 
some  time  the  animal  is  fastened  to  a  board  with  stout  cords,  -01 
is  held  by  means  of  a  specially  constructed  animal  holder. 

Inoculation. — Infectious  material  may  be  introduced  into 
the  animal  body  in  various  ways.  The  most  common  methods 
are  injection  under  the  skin  and  injection  into  the  peritoneal 
cavity.  The  hair  should  be  removed  from  the  site  selected. 
A  sterilized  hypodermic  syringe  is  used,  and  it  is  again 
sterilized  by  boiling  after  use.  Subcutaneous  injection  is 
usually  made  in  the  thoracic  region  as  one  easily  avoids  pene- 
trating the  chest  cavity.  For  intraperitoneal  injection  the 
needle  is  quickly  thrust  through  the  abdominal  wall. 

Inoculation  into  the  cranial  cavity  is  practised  especially  in 
studying  rabies.  The  animal,  rabbit  or  guinea-pig,  is  anesthe- 
tized and  the  scalp  is  shaved.  An  incision  through  the  scalp 
about  8  to  10  mm.  long  is  made  at  the  left  of  the  median  line 
and  parallel  with  it,  a  little  in  front  of  a  line  connecting  the 
external  auditory  openings.  The  scalp  i-s  then  forcibly  drawn 
over  to  the  right  and  a  hole  drilled  though  the  skull  at  the  right  of 
the  median  line.  A  sharp-pointed  scalpel  may  serve  the  purpose 
of  a  drill.  The  needle  is  then  inserted  into  the  cerebral  substance 
nearly  to  the  floor  of  the  cranial  cavity  and  the  material  (o.i 
to  0.5  c.c.)  injected.  Any  blood  or  fluid  is  taken  up  with  sterile 
absorbent  cotton.  The  skin  is  replaced  in  its  original  position 
and  may  be  dressed  with  cotton  and  collodion,  although  dressing 
may  be  omitted  altogether. 

Inoculation  into  the  circulating  blood  is  a  method  of  special 
importance.  In  rabbits  intravenous  injection  is  easily  done. 
The  hair  is  removed  from  the  ear  over  the  marginal  vein,  and 
the  vein  is  dilated  by  application  of  a  hot  towel,  after  which  the 
skin  is  wiped  dry.  An  assistant  constricts  the  base  of  the  ear 
to  congest  the  vein  and  the  needle  is  easily  inserted  into  it. 
Other  veins  on  the  ear  may  be  used,  but  they  are  not  so  easily 
penetrated  by  the  needle.  In  rats,  guinea-pigs  or  monkeys, 
intravenous  injection  is  not  so  simple  and  it  is  easier  to  inoculate 


134  BACTERIOLOGY 

these  animals  by  intracardiac  injection.  For  this  purpose  the 
animal  is  etherized  and  the  precordial  region  is  shaved  and  dis- 
infected. The  material  to  be  injected  is  taken  up  into  a  Luer 
glass  syringe.  A  second  syringe,  empty,  with  needle  attached, 
is  used  to  puncture  the  chest  wall  and  the  heart,  preferably  the 
wall  of  the  right  ventricle.  The  needle  is  introduced  in  the  inter- 
costal space  directly  over  the  heart  and  near  the  border  of  the 
sternum.  The  appearance  of  blood  in  the  previously  empty 
syringe  gives  notice  that  the  cavity  of  the  heart  has  been  entered. 
The  syringe  is  now  detached  from  the  needle  and  the  other 
syringe  which  contains  the  material  to  be  injected  is  quickly 
substituted  for  it.  The  injection  is  made  slowly. 

Other  Sites  for  Inoculation. — Many  other  regions  are  easily 
reached  with  the  injection  needle,  such  as  the  pleural  cavity,  the 
chambers  of  the  eye,  the  spinal  canal,  the  interior  of  muscles, 
and  the  substance  of  the  testis. 

Subcutaneous  Application. — Inoculation  may  be  accomplished 
without  using  a  syringe  if  desired.  The  skin  and  mucous  mem- 
branes may  be  scratched  with  a  needle  or  other  instrument  and 
the  infectious  material  applied  to  the  slight  wound  thus  made. 
A  small  pocket  may  be  made  under  the  skin  by  making  a  small 
incision  and  introducing  a  blade  of  the  forceps  to  separate  the 
skin  from  the  underlying  muscle;  and  into  such  a  pocket  one  may 
introduce  solid  material,  bacteria  from  a  culture,  pieces  of  tissue, 
garden  soil  or  splinters  of  wood,  ^with  accompanying  bacteria. 
The  opening  of  the  pocket  is  closed  by  cauterization  or  sealed 
with  collodion 

Alimentary  and  Respiratory  Infection. — Animals  are  some- 
times infected  by  feeding  the  virus,  occasionally  by  injection 
into  the  rectum.  Infection  of  the  respiratory  tract  by  spraying 
infectious  material^m  the  air  breathed  by  the  animal  is  rarely 
employed. 

Collodion  Capsules. — Bacteria  may  be  cultivated  in  the 
living  body  of  an  animal,  without  infecting  the  animal,  when  they 
are  enclosed  in  collodion  capsules.  Their  soluble  products  are 


METHODS    OF    ANIMAL    EXPERIMENTATION  135 

able  to  diffuse  through  the  collodion,  while  the  animal's  fluids  may 
pass  into  the  sac  to  nourish  them.  These  capsules  were  originally 
made  by  dipping  the  round  end  of  a  glass  rod  into  collodion 
repeatedly.  McCrae's  method1  is  easier  and  more  satisfactory. 
(Fig.  58.) 

A  piece  of  glass  tubing  is  taken,  and  a  narrow  neck  drawn  on  it  near  one 
end.  This  end  of  the  tube  is  rounded  in  the  flame  and,  while  still  warm,  the 
body  of  a  gelatin  capsule  is  fitted  over  it,  so  that  the  gelatin  may  adhere  to 
the  glass.  The  capsule  is  now  dipped  into  3  per  cent  collodion,  covering 
the  gelatin  and  part  of  the  glass.  It  is  allowed  to  dry  a  few  minutes,  and  is 
dipped  again.  In  all,  two  or  three  coatings  may  be  given.  The  capsule  is 
filled  with  water  and  boiled  in  a  test-tube  with  water.  The  melted  gelatin  is 
removed  from  the  inside  of  the  capsule  by  means  of  a  fine  pipette.  The  cap- 
sule is  partly  filled  with  water  or  broth  and  sterilized.  The  capsule  may  now 
be  inoculated.  The  narrow  part  of  the  glass  tube  which  constitutes  the  neck 
must  then  be  sealed  in  the  flame,  taking  care  that  the  neck  be  dry.  The 


(1 


FIG.  58.  —  Method  of  making  collodion  capsules.     (After  McCra.) 

sealed  capsule  should  be  placed  in  bouillon  for  twenty-four  hours.  No 
growth  should  occur  outside  the  capsule  if  it  is  tight.  It  may  now  be  placed 
in  the  peritoneal  cavity  of  an  animal. 

A  method  for  making  collodion  sacs  recommended  by  Gorsline2  consists 
in  the  use  of  a  glass  tube,  the  lower  end  of  which  is  rounded  and  closed, 
except  a  small  hole,  which  is  temporarily  filled  with  collodion.  This  tube  is 
dipped  in  collodion  and  dried,  as  above.  It  may  now  be  filled  with  water. 
By  blowing  at  the  opposite  end,  the  pressure  through  the  hole  in  the  bottom 
of  the  glass  tube  will  cause  the  capsule  to  loosen  so  that  it  comes  away  easily. 
Sacs  made  in  this  way  are  soaked  in  water  for  30  minutes,  dried  and  attached 
to  the  glass  tube  by  gentle  heat.  The  joint  is  wound  with  silk  thread  and 
coated  with  collodion.  The  sac  is  then  filled  with  distilled  water,  immersed 
in  a  tube  of  water  and  sterilized  in  the  autoclave. 

There  are  also  various  other  methods  recommended  for  making  collodion 
sacs. 

1  Journal  of  Experimental  Medicine.     Vol.  VI,  p.  635. 

2  Contributions  to  Medical  Research.     Dedicated  to  Victor  C.  Vaughan,  Ann 
Arbor,  1903,  p.  390. 


136  BACTERIOLOGY 

Collodion  capsules  are  ordinarily  placed  free  in  the  peritoneal 
cavity  of  the  animal,  by  an  aseptic  laparotomy.  The  wound  is 
sutured  with  silk  or  catgut  and  dressed  with  sterile  cotton  and 
collodion. 

Observation  of  Infected  Animals. — In  nearly  every  case  it 
will  be  well  to  keep  a  record  of  the  weight  of  the  animal  from  time 
to  time.  The  temperature  may  be  observed  by  means  of  a 
thermometer  in  the  rectum.  It  should  be  inserted  a  considerable 
distance,  4  to  8  centimeters  in  guinea-pigs.  Other  examinations 
are  made  in  special  cases,  such  as  palpation  of  the  lymph  glands 
in  tuberculosis  and  microscopic  examination  of  the  blood  in  an- 
thrax, trypanosomiasis  and  the  relapsing  spirochetoses. 

The  post-mortem  examination  of  experimental  animals  has 
been  discussed  (pages  98  and  100). 


PART  II. 

GENERAL  BIOLOGY  OF  MICRO- 
ORGANISMS. 


CHAPTER  VII. 
MORPHOLOGY  AND  CLASSIFICATION. 

The  minute  living  things  included  under  the  general  term 
microbe,  are  exceedingly  various  in  form  and  structure  as  well  as 
in  respect  to  food  requirements  and  physiological  activity. 
The  number  of  different  microbes  is  so  great  and  so  great  are 
the  difficulties  involved  in  the  accurate  observation  of  their 
various  features,  that  the  biological  relationships  of  many  of 
the  various  forms  to  each  other  are  not  yet  determined,  and 
much  of  the  generic  and  specific  terminology  in  common  use 
rests  upon  insecure  foundation.  Nevertheless  a  certain  kind 
of  order  has  developed  in  our  conceptions  of  the  grouping  of 
micro-organisms. 

Molds. — The  molds  or  hyphomycetes  are  multicellular  or- 
ganisms characterized  by  the  formation  of  a  network  (mycelium) 
made  up  of  branching  threads  (hypha),  and  by  their  special 
fruiting  organs.  These  threads  vary  from  2  to  7;*  in  width. 
Within  the  group  of  molds  the  structure  of  fruiting  organs  is  used 
as  the  most  important  character  from  which  to  determine  relation- 
ships. The  phycomycetes,  or  algo-fungi,  are  characterized 
by  the  presence  of  sexual  reproduction  in  which  the  union  of 
two  cells  gives  rise  to  resting  cells,  zygospores  and  oospores, 
which  are  enclosed  in  a  thick  wall.  The  ascomycetes  are  char- 

137 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


FIG.  59. — Common  Molds. 

a.  Penicillium    glaucum.     b.  Oidium   lactis.    c.  Aspergillus   glaucus.     d.  The 
same  more  highly  magnified,    e.  Mucor  mucedo.     (Baumgarten.) 


MORPHOLOGY  AND   CLASSIFICATION  139 

acterized  by  the  occurrence  of  a  spore-sac  called  the  ascus  which 
usually  contains  eight  spores.  The  common  aspergilli  belong  here. 
The  basidiomycetes  are  characterized  by  the  occurrence  of  a 
spore-bearing  cell,  the  basidium,  which  bears  four  protuberances 
called  sterigmata  (singular  sterigma)  upon  each  of  which  is  a 
single  spore.  Mushrooms  and  puff-balls  belong  to  this  group. 
Besides  these  three  well-defined  families,  there  are  many  kinds 
of  molds  and  fungi  concerning  which  definite  knowledge's  still 


F-Vy 


FIG.  60. — Yeast  cells  stained  with  fuchsin.     (Xiooo.) 

too  incomplete  for  them  to  be  finally  placed.  The  common 
oidium  and  penicillium  and  many  parasitic  molds  are  included 
here.  The  molds1  are  especially  important  as  causes  of  disease 
in  plants.  Relatively  few  diseases  of  man  or  other  animals  have 
been  shown  to  be  due  to  them,  although  the  first  diseases  proven 
to  be  due  to  micro-organisms  were  those  caused  by  certain  molds. 
The  molds  possess  the  general  morphological  features  of  plants 
except  for  the  absence  of  chlorophyll. 

1  For  fuller  discussion  of  molds  in  general  see  Marshall,  Microbiology,  pp.  12-27, 
article  by  Thorn. 


140 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


Yeasts. — The  yeasts  (Blastomycetes)  are  very  closely  related 
to  the  molds.  In  fact  some  stages  in  the  growth  of  molds  resemble 
very  closely  the  normal  development  of  a  yeast.  The  yeasts, 
however,  do  not  grow  out  into  long  filaments  but  remain  spherical 
or  ovoid.  The  cells  vary  from  2.5  to  12 ft  in  diameter.  During 
active  growth  they  reproduce  by  budding,  a  smaller  portion  being 


FIG.  61. — Wine  and  beer  yeasts.  A,  S.  ellipsoideus,  young  and  vigorous;  B,  S. 
ellipsoideus,  (i)  old,  (2)  dead;  C.  S.  cerevisia,  bottom  yeast;  D,  S.  cerevisice,  top 
yeast.  (After  Marshall.) 

pinched  off  from  the  parent  cell.  The  true  yeasts  also  form  spores 
inside  the  cell,  from  four  to  eight  typical  ascospores,  showing 
their  very  close  relationship  to  molds.  Yeasts  are  very  important 
in  the  fermentation  industries.  Very  few  of  them  are  pathogenic. 
Among  themselves,  the  yeasts  are  subdivided  into  two  groups, 
(i)  those  which  produce  ascospores  (saccharomycetes  or  true 


MORPHOLOGY  AND   CLASSIFICATION  141 

yeasts)  and  (2)  those  which  fail  to  produce  such  spores  (torula 
or  wild  yeasts).  They  are  further  distinguished  by  differences 
in  the  form  of  the  cells,  but  especially  by  differences  in  physi- 
ological characters,  such  as  the  fermentation  of  sugars  and  the 
production  of  pigments. 

In  the  yeasts  there  is  no  definite  differentiation  of  cells. 
Various  cell  structures  such  as  cell-wall,  nucleus  and  cystoplasm 
with  vacuoles  and  granules,  can  be  demonstrated.  The  cell 
membrane  is,  as  a  rule,  more  delicate  than  in  the  molds.  It 
sometimes  secretes  a  gelatinous  material  which  forms  a  thick 
capsule  about  the  cell.  The  nucleus  is  shown  by  appropriate 
methods  of  staining  as  a  single  more  or  less  sharply  defined 
mass  of  chromatin.  Under  suitable  conditions  the  true  yeasts 
produce  endospores,  usually  multiple,  and  as  many  as  eight  in 
one  cell.  These  are  spherical  or  ovoid  masses  surrounded  by  a 
definite  wall,  and  usually  about  half  the  diameter  of  the  yeast 
cell.  When  supplied  with  nutriment  these  spores  swell  and  burst 
the  mother  cell,  and  then  begin  at  once  to  multiply  by  budding. 
Dry  commercial  yeast  cakes  contain  spores  of  yeast  along  with 
bacteriaand  molds;  moist,  "compressed,"  yeast  contains  vegetat- 
ing yeast  cells,  also  mixed  with  other  organisms. 

Bacteria. — Bacteria  (schizomycetes)  are  minute  unicellular 
organisms  0.2  to  4//.  in  width  which  multiply  solely  by  simple 
transverse  division  (fission),  ordinarily  resulting  in  the  produc- 
tion of  two  cells  of  equal  size.  In  many  instances  the  cells  re- 
main attached  to  each  other  so  as  to*  form  long  filaments. 

Trichobacteria. — Certain  of  them  grow  into  long  filaments 
without  dividing  at  once  into  shorter  segments.  These  forms 
which  are  classed  as  higher  bacteria  or  trichobacteria,  suggest 
a  very  close  relationship  to  the  molds  and  may,  perhaps,  be  re- 
garded as  intermediate  between  the  molds  and  the  lower  bacteria. 
Many  of  them  exhibit  a  differentiation  of  the  filament  into  base 
and  apex,  some  of  them  branch  in  an  irregular  fashion,  and  in 
some  there  is  a  suggestion  of  the  formation  of  special  fruit 
organs.  These  higher  bacteria  require  further  study  to  deter- 


142  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

mine  their  relationships.  A  few  of  them  are  important  patho- 
genic agents. 

The  Lower  Bacteria. — The  lower  bacteria,  or  true  bacteria, 
are  always  simple  in  form,  the  transverse  division  producing 
cells,  relatively  short,  and  of  nearly  equal  length.  Long  filaments 
are  produced  only  by  the  attachment  of  many  individual  cells 
together,  end  to  end.  There  are  no  special  fruit  organs.  The 
special  resistant  form,  or  spore,  which  occurs  in  some  forms  is 
produced  only  inside  of  the  vegetative  cell,  one  cell  producing 
one  spore.  There  are  three  general  forms  of  bacteria,  the  sphere 
(coccus,  plural  cocci),  the  cylinder  (bacillus,  plural  bacilli),  and 
the  spiral  or  segment  of  a  spiral  (spirillum,  plural  spirilla).  In- 
termediate forms  occur,  so  that  there  is  not  a  sharp  line  between 
the  groups.  These  three  forms  are  generally  accepted  as  a  basis 
for  division  of  the  lower  bacteria  into  three  families,  the  coccaceae, 
bacteriaceae  and  the  spirillaceae. 

Spherical  Bacteria. — The  Coccacea  or  cocci  are  spherical 
bacteria.  They  vary  in  size  from  about  0.3^  to  3^  in  diameter. 


Staphylococci.     Streptococci.     Diplococci.     Tetrads.        Sarcinae. 

FIG.  62. 

During  the  process  of  cell  division,  a  coccus  may  become  elongated 
somewhat,  and  after  division,  the  daughter  cells  may  be  shortened 
so  that  they  appear  as  if  compressed  against  each  other.  Slightly 
elongated  forms  are  included  among  the  cocci  in  certain  instances, 
and  especially  the  lancet-shaped  bacteria  such  as  the  germ  of 
lobar  pneumonia.  The  recognition  of  genera  within  the  family 
is  still  unsettled.  Morphologically  five  genera  have  been  dis- 
tinguished by  Migula:  Streptococcus,  Micrococcus,  Sarcina,  Piano- 
coccus  and  Planosarcina.  The  first  three  do  not  possess  flagella 
and  are  non-motile.  Streptococcus  includes  those  forms  which 
divide  only  in  one  plane  so  that  a  thread  or  chain  is  produced. 
Micrococcus  includes  the  cocci  which  divide  in  two  planes  at 


MORPHOLOGY  AND    CLASSIFICATION  143 

right  angles  so  as  to  produce  plates,  and  it  also  includes  those 
which  divide  in  an  irregular  fashion  so  that  no  definite  geometric 
figure  results.  Sarcina  includes  those  cocci  which  divide  in  three 
planes  at  right  angles  to  each  other,  in  turn,  so  as  to  produce 
cubical  masses  of  cells.  Planococcus  is  similar  to  Micrococcus 
in  all  respects  except  that  its  members  are  motile  and  possess 
flagella,  and  Planosarcina  includes  the  motile  forms  which  are 
in  other  respects  the  same  as  the  forms  included  under  Sarcina. 

COCCACE^E— Cells  spherical,  without  endospores. 
Streptococcus — Division  in  one  plane,  forming  chains  of  cells; 

non-motile;  without  flagella. 
Micrococcus — Division  in  two  planes,  forming  flat  plates  of 

cells,  or  irregular,  forming  masses  of  cells  irregularly 

grouped;  non-motile;  without  flagella. 

Sarcina — Division  in  three  planes,  forming  cubical  or  package- 
shaped  masses  of  cells;  non-motile;  without  flagella. 
Planococcus — Division  in  two  planes,   forming  flat  plates 

of  cells,  or  irregular,  forming  mass  of  cells  irregularly 

grouped;  motile;  bear  flagella. 
Planosarcina — Division   in    three    planes,    forming    cubical 

or  package-shaped  masses  of  cells;  motile;  bear  flagella. 
These  genera  have  not  been  generally  adopted  by  bacteriolo- 
gists. The  terms  Streptococcus  and  Sarcina  are,  however, 
quite  generally  employed  as  the  generic  names  for  the  organisms 
of  their  respective  groups  as  defined  by  Migula,  as  they  had 
been  used  in  this  way  before.  Micrococcus,  however,  is  commonly 
employed  as  a  general  term  for  all  the  members  of  the  family 
Coccaceae,  and  Planococcus  and  Planosarcina  have  not  been  used, 
because  bacterial  forms  belonging  to  these  genera  are  exceedingly 
uncommon  and  it  may  even  be  questioned  whether  those  which 
have  been  described  might  not  better  be  classed  with  the  cylin- 
drical bacteria,  in  which  motility  is  of  frequent  occurrence. 
Other  terms  in  common  use  as  generic  names  for  certain  cocci 
are  Diplococcus  and  Staphylococcus.  A  diplococcus  is  a  double 
coccus,  two  spheres  attached  together.  This  grouping  by  twos 


144  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

is  very  common  and  the  generic  term  Diplococcus  is  employed 
for  those  forms  in  which  it  is  a  prominent  characteristic.  The 
term  Staphylococcus  is  applied  to  those  micrococci  which  are 
grouped  in  an  irregular  mass  resembling  a  bunch  of  grapes. 

Cylindrical  Bacteria. — The  cylindrical  bacteria,  Bacteriacece, 
have  been  subdivided  by  Migula  into  three  genera,  Bacterium, 
Bacillus  and  Pseudomonas.  The  genus  Bacterium  includes 
those  members  of  the  family  which  are  without  flagella  and 
are  non-motile.  Bacillus  includes  those  forms  possessing  flagella 
distributed  over  the  surface,  and  Pseudomonas  is  the  generic 
term  for  those  forms  with  flagella  situated  at  the  extremities 
only  (polar  flagella). 

BACTERIACE^E— Cells    cylindrical,    straight,     non- 
motile  or  motile  by  means  of  flagella. 

Bacterium — Cells  without  flagella,  non-motile. 

Bacillus — Cells  motile   with    flagella    distributed   over  the 
surface. 

Pseudomonas — Cells  motile  with  polar  flagella. 
These  genera  have  not  been  generally  adopted  by  bacteri- 
ologists, and  there  are  serious  reasons  for  dissatisfaction  with 
such  a  classification  of  the  rod-shaped  bacteria.  In  the  first 
place  the  names  Bacterium  and  Bacillus  are  unfortunate.  The 
former  has  long  been  employed  as  a  general  term  designating 
any  member  of  the  Schizomycetes  and  its  plural,  Bacteria, 
is  everywhere  the  common  term  employed  in  designating  this 
large  group  of  micro-organisms.  Its  use  in  the  narrower  sense 
by  Migula  has  not  displaced  the  former,  signification,  and  its 
use  in  the  sense  of  Migula  must  necessarily  result  in  confusion. 
The  latter  term,  Bacillus,  has  long  been  used  very  generally  by 
bacteriologists  to  designate  any  member  of  the  Bacteriaceae 
or  rod-shaped  bacteria,  regardless  of  the  motility  or  distribution 
of  flagella.  A  further  serious  objection  is  due  to  the  lack  of 
stability  in  the  character  selected  to  distinguish  the  genera. 
The  flagella  may  disappear  from  bacteria  ordinarily  possessing 
them  as  a  result  of  changes  in  environment  and  may  be  again 


MORPHOLOGY   AND    CLASSIFICATION  145 

made  to  appear  by  reversing  the  conditions.1  Furthermore 
in  some  groups  of  bacteria  which  seem  to  be  closely  related  in 
respect  to  other  characters,  morphological  and  physiological, 
both  motile  and  non-motile  forms  occur.  On  the  whole  the  pres- 
ence or  absence  of  flagella  would  seem  to  be  too  fragile  a  character 
to  serve  as  a  sole  distinction  between  genera  among  the  rod- 
shaped  bacteria. 


FIG.  63. — Bacilli  of  various  forms. 

The  different  species  of  rod-shaped  bacteria  are  very  numerous, 
several  thousand  different  kinds  having  been  described.  They 
vary  in  width  from  4/1  to.  o.i  or  probably  less,  and  in  length  from 
from6o,«to  o.2//.  The  very  large  ones  are  non-pathogenic  species. 
The  form  is  ordinarily  that  of  a  straight  cylinder  of  equal  caliber 
throughout  its  length.  Certain  slightly  curved  forms  are  never- 
theless included  in  the  family,  although  they  may  perhaps  be 
regarded  as  intermediate  between  the  bacteriaceae  and  the 


CMD    CMD 


FIG.  64.  —  Sporulation.     a,  First  stage  showing  sporogenic  granules;  b,  incomplete 
spore;  c,  fully  developed  spore.     (After  Novy.) 

spirillaceae.  Some  of  the  rod-shaped  bacteria  are  of  uneven 
caliber,  especially  when  growing  under  unfavorable  conditions  or 
when  spores  are  produced.  The  ends  of  the  rod  may  be  pointed, 
rounded,  square-cut  or  concave.  The  bacteria  may  remain 
attached  after  cell-division,  forming  groups  of  two,  diplo-bacillus  , 
or  many  cells  remain  attached,  to  form  long  threads,  strepto- 
bacillus.  Endospore  formation  occurs  almost  exclusively  in 

1Passini:  Zts.f.  Hyg.,  1905,  XLIX,  pp.  135-160. 


146  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

the  bacteriaceae  and  the  form  of  the  spore-bearing  cell  differs 
for  different  species  and  is  fairly  constant  for  any  one  species. 


FIG.  65. — Position  of  spores;  resultant  forms  (diagrammatic),  a,  Median 
spores;  b,  intermediate  spores;  c,  terminal  spores;  20,,  b,  c,  change  in  form  of  cells 
due  to  the  presence  of  the  spore;  20,  clostridium;  20,  drum-stick  form.  (After  Novy.} 

The  spore,  which  is  always  single,  may  be  located  at  the  center  of 
the  cell,  median  spore,  or  at  the  end,  terminal  spore,  or 
at  an  intermediate  point.  The  spore-bearing  cell  may  retain 
its  normal  outline  or  it  may  be  bulged  by  the  spore.  The  cell 
containing  a  median  spore  with  bulging  is  called  a  clostridium; 
one  with  terminal  spore  with  enlargement  of  the  cell  is  spoken 
of  as  a  drumstick  or  sometimes  as  a  plectridium. 

Spiral  Bacteria. — The  screw-shaped  bacteria,  Spirillacea,  have 
been  subdivided  into  four  genera  by  Migula.  The  genus  Spiro- 
soma  includes  those  spirals  which  are  rigid  and  without  motility. 
Motile  cells  possessing  one,  two  or  three  polar  flagella  are  classes 
in  the  genus  Microspira;  while  those  possessing  more  than  three 
are  put  in  the  genus  Spirillum.  The  genus  Spirochaeta  includes 
the  slender  flexuous  forms  of  spirals. 

SPIRILLACE^E— Cells   circular   in    cross-section   but 

curved  to  form  a  spiral  or  segment  of  a  spiral. 
Spirosoma — Cells  rigid,  without  flagella,  motionless. 
Microspira — Cells  rigid,  motile,  with  i  to  3  polar  flagella. 
Spirillum — Cells  rigid,  motile,  with  polar  tufts  of  flagella. 
Spirochaeta — Cells  slenders,  flexuous,  motile. 
Two  of  these  generic  terms,  Spirillum  and  Spirochaeta,  have 
long  been  used,  and  almost  in  the  sense  in  which  they  are  em- 
ployed by  Migula.     Spirillum  has  frequently  been  applied  to 


MORPHOLOGY  AND    CLASSIFICATION  147 

all  the  Spirillaceae  and  especially  to  those  forms  which  Migula 
includes  in  his  first  three  genera,  Spirosoma,  Microspira  ancf 
Spirillum.  The  distinction  between  Microspora  and  Spirillum 
seems  of  too  slight  importance  to  serve  as  a  basis  for  the  formation 
of  two  genera,  and  indeed  the  same  objection  exists  here  as  in 
the  Bacteriaceae  to  the  use  of  flagella  as  a  basis  for  generic 
distinctions. 

Cell  division  occurs  by  simple  transverse  fisson  in  all  the  spiral 
bacteria.  Endospores  are 

said  to  be  formed  by  some  .     / 

species.  $fr     ,Tu    ^   \f 

The   group    of     spiro-     V 
chaetes  has  received  much  O/V^W^ 

attention  during  the  past  FlG.  66.— Types  of  spirilla. 

decade  and  the  propriety 

of  including  them  in  the  spirillaceae  may  be  seriously  questioned. 
Many  investigators  are  inclined  to  regard  them  as  more  properly 
classed  with  the  protozoa  than  with  the  bacteria.  It  is  claimed 
that  these  forms  multiply  by  longitudinal  splitting  and  not  by 
transverse  fission,  and  this  would  at  once  remove  them  from  the 
Schizomycetes.  The  observations  are  still  in  dispute  and  there 
are  good  observers  who  regard  transverse  fisson  as  the  mode  of 
multiplication.  Further  study  is  necessary  to  settle  this  impor- 
tant question.  It  is  possible  that  some  of  these  slender  spirals 
may  multiply  by  both  methods,  or  that  one  species  may  divide 
longitudinally  and  another  transversely,  but  this  does  not  seem 
probable.  For  the  present  it  would  seem  wise  to  reserve  judg- 
ment and  avoid  encumbering  the  group  with  new  genera  until  a 
definite'  and  final  agreement  has  been  reached  concerning  the 
exact  morphological  facts.  (See  page  353.) 

Structure  of  Lower  Bacteria. — The  bacterial  cell  is  enclosed 
in  a  relatively  stiff  cell  membrane,  which  generally  retains  its  form 
after  plasmolysis.  Under  special  conditions  of  growth  many 
forms  of  bacteria  become  enclosed  in  a  gelatinous  capsule.  This 
seems  to  be  a  viscid  material  secreted  by  the  cell  through  the  cell 


148  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

membrane.     The  motile  bacteria  possess  exceedingly  slender  hair- 
like  processes,  called  flagella,  which  serve  as  organs  of  locomotion. 
These  processes  apparently  take  origin  from  the  cell  membrane. 
Bacteria  without  flagella  are  spoken  of  as  atrichous,  those  with  a 
single  flagellum  at  one  end  as  monotrichous,  those  with  a  flagellum 
at  either  end  as  amphitrichous.     When  there 
^  ft         is  a  tuft  of  flagella  at  the  end,  the  distribution 

®  ($)  ^  /^  K  is  said  lobe lophotrichous,  and  when  they  are 
distributed  all  over  the  surface  the  arrange- 
ment is  called  peritrichous.  The  internal 


structure  of  the  bacterial  cell  has  received 
FIG.  67. — Bactena  with 

capsules.  comparatively  little  attention.     The   direct 

microscopic  study  of  the  living  cells  shows 
them  to  be  finely  or  coarsely  granular,  or  sometimes  nearly  ho- 
mogeneous. No  constant  internal  structures  can  be  distinguished. 
Ordinary  simple  staining  w'th  the  basic  aniline  dyes  colors  the  bac- 
terial cell  diffusely  and  intensely,  usually  without  any  internal 
differentiation.  The  cell  membrane  between  two  cells  in  a  chain 
may  remain  relatively  colorless  and  so  be  differentiated  from  the 
protoplasm  on  either  side.  At  times  the  stainable  substance  is  un- 
evenly distributed  in  the  cell,  perhaps  grouped  at  the  ends  of  a  rod, 


FIG.  68. — Bacteria  showing  flagella. 

or  in  granules  or  bands.  Under  special  conditions  some  bacteria 
show  internal  granules  of  special  composition,  distinguishable  as 
pigment  granules  or  by  their  microchemical  reactions.  Granules 
which  stain  with  iodine,  so-called  granulose  or  glycogen  granules, 
are  important  features  of  some  kinds  of  bacteria. 

The  recognition  of  the  cell  nucleus  has  received  special  atten- 
tion. Zettnow,  more  especially,  has  shown  that  the  chromatin  or 
essential  nuclear  substance  is  present  in  the  bacterial  cell  as  finer 


MORPHOLOGY  AND    CLASSIFICATION  149 

or  larger  granules,  sometimes  distributed  pretty  generally  and 
sometimes  collected  together  at  one  or  more  places  in  the  £elL 
The  Romanowsky  stain  and  its  modifica- 
tions have  been  especially  useful  in  differ- 
entiation of  chromatin  from  cytoplasm. 

Special  movements  of  the  internal 
granules  have  been  described  by  Schau- 
dinn  as  being  associated  with  beginning 

cell  division.     For  the  great  majority  of       FIG.  69. — The  formation  of 
,-,  ,     spores.     (After    Fischer   from 

bacteria  these  have  not  been  observed,  frost  and  McCampbdl.) 

and    according   to    our    knowledge,   the 

process  of  cell  division  is  extremely  simple.     It  consists  of  a  pro- 
gressive constriction  and  thinning  of  the  cell  at  the  middle  until 


FIG.  70. — Bacteria  with  spores. 

two  cells  are  produced.     In  some  forms  the  division  is  completed 
by  a  sudden  snapping  movement. 

The  formation  of  an  endospore  begins  with  the  accumulation 

o  0  C 


n 
u 


d 


FIG.  71. — Germination  of  spores,  a,  Direct  conversion  of  a  spore  into  a  bacillus 
without  the  shedding  of  a  spore- wall  (B.  leptosporus);  b,  polar  germination  of  B. 
anthracis,  c,  equatorial  germination  of  B.  subtilis;  d,  same  of  B.  megatherium;  „, 
same  with  "horse-shoe"  presentation.  (After  Novy.) 

of  chromatin  granules  in  one  part  of  the  cell,  where  they  coalesce, 
lose  their  contained  water  and  seem  to  become  embedded  in  an  oily 


150  GENERAL  BIOLOGY   OF  MICRO-ORGANISMS 

or  fatty  substance  and  surrounded  by  a  membrane.  Very  early  in 
the  process  the  spore  no  longer  stains  readily.  In  some  forms 
(Bact.  anthracis)  the  cell  in  which  a  spore  has  formed  disintegrates 
rapidly,  setting  free  the  spore,  while  in  others  (B.  telani)  the  cell 
may  continue  its  activities  after  formation  of  the  spore.  The  spore 
germinates  when  conditions  again  become  favorable  to  active 
growth.  The  new  cell  may  burst  the  spore  wall  into  halves,  or 
at  the  end,  or  the  spore  wall  may  soften  and  become  a  part  of  the 
new  growing  cell. 

Filterable  Viruses. — The  difficulty  of  accurate  morphological 
study  is  so  great  as  to  appear  insurmountable  in  the  case  of  cer- 
tain microbes  which  are  very  definitely  recognizable  by  certain 
effects  which  they  produce.  This  is  especially  true  of  those 
living  things  capable  of  passing  through  the  fine  filters  which 
prevent  the  passage  of  small  bacteria.  The  causes  of  certain 
diseases  exhibit  this  character,  and  these  have  come  to  be  known 
as  filterable  viruses.  There  can  be  little  question  that  non-patho- 
genic filterable  microbes  also  exist  although  they  seem  to  have 
escaped  observation.  Accurate  knowledge  of  the  morphology 
of  these  forms  remains  to  be  disclosed  by  future  investigation. 
Meanwhile,  the  efforts  to  classify  them  as  bacteria  or  as  protozoa 
may  well  be  spared.  The  propriety  of  including  them  as  living 
things  is,  however,  only  occasionally  questioned. 

Protozoa. — The  protozoa  or  unicellular  animals  have  assumed 
very  great  importance  as  causes  of  disease  during  the  past  dec- 
ade. Fortunately  for  the  systematist,  the  free-living  protozoa 
had  received  considerable  careful  study  and  the  larger  groups  of 
protozoa  had  been  well  defined  before  the  interest  in  pathogenic 
properties  had  the  opportunity  to  over-shadow  morphological 
study.  The  number  and  variety  of  easily  recognizable  morpho- 
logical characters  presented  by  the  protozoa  are  greater  than 
those  of  the  bacteria;  and  the  organisms  are,  on  the  whole, 
larger.  These  factors  make  for  more  accurate  observations  of 
morphological  characters,  and  their  more  successful  employment 
as  a  basis  of  classification. 


MORPHOLOGY   AND    CLASSIFICATION  151 

The  protozoan  cell  is  generally  larger  and  more  complex  in 
structure  than  the  bacterial  cell  appears  to  be,  although  the 
viding  line  is  in  places  indefinite  or  even  wholly  obscure.  In 
general  the  protozoon  shows  the  typical  structure  of  a  single  cell 
of  the  metazoon.  A  well-defined  nucleus  is  usually  present,  some- 
times several  of  them,  although  in  some  forms  the  nuclear  ma- 
terial is  more  or  less  scattered  throughout  the  cell.  Most  proto- 
zoa exhibit  differentiation  of  the  protoplasm  into  cell  organs  or 
organellae,  adapted  to  perform  certain  functions.  In  many  pro- 
tozoa sexual  reproduction  has  been  observed,  a  process  involving 
complex  morphological  changes.  The  cells  showing  these  evi- 
dences of  complex  organization  resemble  in  most  respects  cells 
of  the  higher  animals,  and  in  fact  a  colony  or  group  of  protozoa 
may  be  regarded  as  representing  a  transition  to  the  many-celled 
animals,  just  as,  on  the  other  hand,  the  bacteria  were  seen  to  be 
connected  with  the  higher  plants  through  the  forms  of  the  higher 
bacteria,  the  yeasts,  the  molds  and  algae.  Physiologically,  pro- 
tozoa differ  from  bacteria  and  other  plants  in  requiring  more  com- 
plex nitrogenous  food,  but  this  distinction  is  far  from  absolute. 
Doflein  divides  the  protozoa  into  two  substems,  (i)  Plasmodroma, 
including  those  forms  which  move  by  means  of  pseudopodia  or 
flagella,  and  which  exhibit  for  the  most  part  an  alternation  of 
asexual  and  sexual  generations,  and  (2)  Ciliophora,  including 
those  forms  which  move  by  means  of  cilia  and  in  which  the  sexual 
fertilization  gives  rise  to  no  special  reproductive  form  of  the 
organism. 

The  substem  Plasmodroma  includes  three  classes,  (i)  Masti- 
gophora,  (2)  Rhizopoda  and  (3)  Sporozoa. 

Flagellates. — In  the  class  Mastigophora,  are  included  a  great 
many  different  organisms,  the  one  common  feature  being  the 
type  of  locomotive  apparatus,  which  consists  of  one  or  more  fla- 
gella. The  further  subdivision  of  the  class  has  not  yet  been  agreed 
upon,  not  because  of  any  lack  of  morphological  differences  upon 
which  to  base  a  classification,  but  largely  on  account  of  difficulty 
in  estimating  the  relative  importance  and  meaning  of  the  many 


152 


GENERAL  BIOLOGY    OF   MICRO-ORGANISMS 


B 


D 


H 


FIG.  72. — The  most  important  trypanosomes  parasitic  in  mammals.  A,  Try- 
panosoma  lewisi  (Kent).  B,  Tr.  evansi  (Steele),  Indian  variety.  C,  Tr.  evansi 
(Steele),  Mauritian  variety.  D,  Tr.  brucei  (Plimmer  and  Bradford).  E,  Tr.  equiper- 
dum  (Doflein).  F,  Tr.  equinum  (Voges).  G,  Tr.  dimorphon  (Laveran  and  Mesnil). 
H,  Tr.  gambiense  (Button).  (From  Doflein  after  micro  photo  graphs  of  Novy.) 


FIG.  73. — Leishmania  donovani.     Various  forms  obtained  by  spleen  puncture,  some 
free  and  some  inside  red  blood  cells.     (From  Doflein  after  Donoian.) 


MORPHOLOGY   AND    CLASSIFICATION 


153 


criteria  presented.  The  genera  of  particular  interest  from  the 
pathological  standpoint  are  Trypanosoma,  Leishmania,,  Tricho- 
monas  and  Lamblia.  The  members  of  the  Trypanosoma  are 


FIG.  74. — Leishmania 
donovani.  Various  forms 
of  the  organism  in  artifi- 
cial culture.  (From  Doflein 
after  Chatterjce.} 


FIG.  75. — Trichomonas     hominis. 
(From  Doflein  after  Grassi.} 


characterized  by  an  approximately  crescent-shaped  body,  10  to 
40ft  in  length,  flexible  and  provided  with  a  flagellum  which  origi- 


FIG.  76. — Lamblia    inttstinalis.     A,  Ventral   aspect.     B,  Lateral   aspect.     C.   At- 
tached to  an  epithelial  cell.         (From  Doflein  after  Grassi  and  Schewiakoff.) 

nates  in  the  endoplasm  near  one  end  and  passes  along  the  border 
of  the  body  and  finally  projects  as  a  free  whip  at  the  other  end 
of  the  cell.  As  it  passes  along  the  border  of  the  cell  it  is  enclosed 


154 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


in  a  sheath  of  ectoplasm,  which  is  drawn  out  into  a  thin  sheet 
forming  an  undulating  membrane.  Multiplication  takes  place 
by  approximately  longitudinal  division.  Leishmania  includes  a 
few  parasitic  forms,  for  the  most  part  living  inside  the  cells  of 
the  host.  These  organisms  are  oval,  about  2X3^,  without  fla- 
gellum  or  undulating  membrane.  In  artificial  culture  outside 
the  body,  the  protozoon  grows  larger,  develops  a  flagellum  and 
resembles  a  trypanosome.  Trichomonas  includes  pear-shaped 


£  F  G  H  I 

FIG.  77. — Entamceba  coli  (Losch).  A  to  C,  Various  forms  of  the  free  ameba. 
D,  Stage  with  eight  nuclei.  £  to  G,  Cysts  with  various  numbers  of  nuclei.  H, 
Opening  cyst.  1,  Young  amebae  escaped  from  a  c>st.  (From  Doflein  after  Casa- 
grandi  and  Barbagallo.) 

organisms  4  to  30/4  in  diameter,  provided  with  three  or  four  fla- 
gella.  Isogamic  and  autogamic  fertilization  have  been  described, 
and  cysts  containing  numerous  daughter  cells  result  from  the 
multiplication  following  this  process.  Lamblia  resembles  tricho- 
monas,  but  the  cell  is  here  shaped  more  like  a  beet,  is  provided 
with  eight  flagella  and  is  hollowed  out  at  one  side  near  the 
rounded  anterior  end  to  form  a  suction  cavity. 

Rhizopods. — The  members  of  the  second  class,  Rhizopoda, 
are  characterized  by  their  ability  to  send  out  protoplasmic  proc- 


MORPHOLOGY  AND    CLASSIFICATION  155 

esses  to  serve  for  locomotion  and  also  to  surround  and  engulf 
solid  food  particles.  The  two  genera,  Amoeba  and  Entamaeba, 
are  of  chief est  interest.  The  organisms  are  masses  of  protoplasm 
containing  a  nucleus,  food  granules  and  sometimes  vacuoles, 
and  surrounded  by  a  slightly  denser  more  hyaline  layer  of  ecto- 
plasm. The  members  of  the  genus  Amoeba  are  free-living 
saprophytic  forms,  while  those  of  Eniamosba  are  parasitic. 
Multiplication  occurs  by  fission  after  a  more  or  less  complex  di- 
vision of  the  nucleus.  Multiple  division  also  occurs,  more  es- 
pecially in  an  encysted  condition,  and  subsequent  to  a  possible 
autogamic  fertilization. 

Sporozoa. — The  third  class,  Sporozoa,  is  made  up  entirely 
of  parasitic  forms,  which  at  some  stage  in  their  life  history  multiply 
by  division  into  numerous  daughter  cells,  which  are  enclosed  in  a 
protective  envelope  to  form  a  spore.  The  spores  serve  to  dis- 
tribute the  species  to  other  hosts.  In  cases  where  there  are  special 
adaptations  for  distribution,  as  for  example  by  means  of  inter- 
mediate hosts,  the  protective  envelope  may  be  absent.  An  enor- 
mous number  of  parasitic  micro-organisms  are  included  in  this 
group.  The  genera  of  greatest  present  interest  from  the  patho- 
logical point  of  view  are  Eimeria  (Coccidium),  Plasmodium 
Babesia  (Piroplasma)  and  Nosema. 

The  Coccidia. — Eimeria  includes  a  number  of  intracellular 
parasitic  forms,  perhaps  better  known  as  coccidia.  The  small 
parasite  resulting  from  asexual  division  is  called  a  merozoit.  It 
is  somewhat  spindle-shaped  and  5  to  IOJJL  long.  This  merozoit 
penetrates  an  epithelial  cell  of  the  host,  grows  at  the  expense  of 
the  cell  to  a  spherical  mass  20  to  50;*  in  diameter,  and  eventually 
divides  into  numerous  (sometimes  as  many  as  200)  merozoits, 
which  become  free  by  rupture  of  the  host  cell.  Besides  this  asexual 
mode  of  multiplication,  there  is  also  a  sexual  cycle.  Some  of  the 
growing  parasites  do  not  divide  into  merozoits  but  become  differ- 
entiated into  male  and  female  cells  (gametocy tes) .  The  male 
gametocyte  gives  rise  to  a  large  number  of  elongated  motile  micro- 
gametes,  one  of  which  approaches  and  penetrates  the  ripened 


156 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


jzr 


FIG.  78. — Developmental  cycle  of  Eimeria  (Coccidium)  schubergi.  I,  Sporzoit; 
II,  sporozoit  penetrating  a  cell  of  the  host;  III  and  IV,  stages  of  growth;  V  to 
VII,  asexual  multiplication;  VIII,  agamete  or  merozoit  beginning  again  the  asexual 
cycle;  IX  and  X,  agametes  destined  to  form  sexual  cells  (gametes);  XI,  a  to  c,  devel- 
opment of  the  macrogamete;  XII,  a  to  d,  development  of  microgametes;  XIII, 
fertilization;  XIV  and  XV,  the  fertilized  cell  or  zygote;  XVI  and  XVII,  metagamic 
division  of  the  zygote;  XVIII,  formation  of  the  sporoblasts;  XIX,  formation  of 
the  spores  and  sporozoits;  XX,  sporozoits  emerging  from  the  spores  and  from  the 
oocyst.  (From  Doflein  after  Schaiidinn,) 


MORPHOLOGY   AND   CLASSIFICATION 


157 


macrogamete.  The  nuclei  of  the  two  gametes  fuse  and  the 
fertilized  cell  quickly  forms  a  protective  wall  around  itself  and  then 
divides  into  eight  cells  which  are  enclosed  in  pairs  within  secondary 
cysts  known  as  spores.  This  form  of  the  organism  passes  out  of  the 
host,  and  after  a  passive  existence  in  the  external  world  may  gain 
entrance  to  a  new  host,  whereupon  the  spore  wall  ruptures  and  the 
enclosed  cells,  sporozoits,  emerge  to  penetrate  new  host  cells. 

The  Plasmodia. — Plasmodium  includes  the  malarial  parasites, 
forms  parasitic  in  red  blood  cells  and  closely  analogous  to  the 


FIG.  79. — Forms  in  the  asexual  cycle  of  Plasmodium  falciparum,  the  parasite 
of  tropical  malaria.  A,  Multiple  infection  of  a  red  blood  cell;  B  to  E,  various  forms 
of  the  growing  parasite;  B  and  C  show  also  the  Maurer  granulations;  F,  full-grown 
parasite  with  many  nuclei;  G,  Segmentation.  The  pigment  is  shown  in  E,  F  and  G. 

(After  Doflein,} 

coccidia  in  the  asexual  cycle.  The  garnet  ocytes  are  also  similar  to 
those  of  Eimeria  except  that  the  gametes  are  not  formed  within 
the  mammalian  host,  but  only  after  the  blood  has  been  drawn. 
The  sexual  cycle  of  development  takes  place  in  a  definite  secondary 
host,  the  mosquito.  In  the  stomach  of  this  insect  the  gametes 
unite  and  the  fertilized  cell  (ookinet)  actively  penetrate?  the 
epithelium  and  beneath  it  develops  into  a  large  oocyst,  30  to  90^ 
in  diameter,  enclosed  in  the  elastic  tunic  of  the  stomach  wall  of  the 
mosquito.  As  the  oocyst  enlarges  the  nucleus  divides  and  eventu- 


158  GENERAL  BIOLOGY  OF  MICRO-ORGANISMS 

ally  the  cytoplasm  also.  The  nucleus  of  each  of  these  masses 
(sporoblasts)  then  divides  many  times.  Each  nucleus,  together 
with  a  small  amount  of  protoplasm,  separates  and  then  elongates 
into  a  slender  thread-like  sporozoit  (14X1/0  •  As  many  as  io,ooc 
of  these  may  be  produced  in  one  oocyst.  The  cyst  bursts  into  the 
body  cavity  of  the  mosquito  and  the  motile  sporozoits  circulate 
through  the  body  of  the  insect  and  eventually  assemble  in  the  cells 
of  the  salivary  glands.  From  these  they  escape  with  the  secretion 
and  gain  entrance  to  the  wound  made  by  the  mosquito  in  biting. 
Babesia. — A  number  of  parasites  of  the  red  blood  cells  are 
classed  in  the  genus  Babesia  (Piro  plasma).  These  resemble  the 
members  of  the  preceding  genus  very  closely  but  multiple  division 
(segmentation)  does  not  seem  to  occur  in  the  asexual  cycle.  The 

v  G^~          H 


FIG.  8c. — Babesia  muris.  A,  Young  form  in  a  red  blood  cell.  B,  Form  with 
two  nuclei.  C  and  D,  Binary  division.  E  and  F,  Multiple  infection;  ameboid 
forms  in  F.  G,  An  exceptionally  large  individual  (gametocyte?).  H,  Form  with  a 
thread-like  process  (flagellated  stage?).  (From  Doflein  after  Fantham.) 

multiplication  seems  to  be  by  longitudinal  division  into  two 
daughter  cells.  The  characteristic  form  is  pear-shaped,  but 
irregular  amoeboid  'forms  are  also  common.  Flagellate  stages 
existing  in  the  blood  plasma  have  also  been  described.  The  sexual 
cycle  takes  place  in  a  tick,  and  is  in  part  analogous  to  that  described 
for  Plasmodium.  The  stages  are  not  fully  known,  but  the  infec- 
tivity  of  the  tick  is  transmitted  to  the  offspring  in  the  case  of  the 
Texas-fever  tick  (Rhipicephalus  (Boophilus)  annulatus). 

Nosema. — The  sporozoa  above  described  all  belong  to  the 
Telosporidia,  organisms  which  end  their  individual  existence  at 
the  stage  of  spore  formation.  A  second  large  subdivision  of  the 
sporozoa  is  named  Neosporidia.  In  this  group  the  spores  are 
formed  without  terminating  the  existence  of  the  individual.  The 


MORPHOLOGY  AND   CLASSIFICATION 

parasites  of  this  type  are  comparatively  small  and  not  very  well 
known.  They  are  often  spoken  of  as  microsporidia  or  psorosperm^, 
The  best-known  form  is  Nosema  bombycis,  the  cause  of  Pebrine  in 
silkworms. 

Ciliates. — The  second  substem  of  the  protozoa,  Ciliophora, 
is  distinguished  by  the  locomotive  organs,  numerous  cilia  which 


FIG.  8 1. — Diagram  of  the  developmental  cycle  of  Nosema  bombycis.  C,  Cell  of 
the  intestinal  epithelium  containing  asexual  multiplication  forms  and  showing  their 
transition  into  spores,  a,  b,  c,  Spores,  the  last  with  polar  thread,  d,  Ameboid  form 
emerging  from  the  spore  to  penetrate  a  new  host  cell  at  h.  (From  Dofiein  after 
Stempell.} 

cover  most  of  the  body  surface,  and  by  the  possession  of  two  dis- 
tinctly different  nuclei,  one  apparently  concerned  with  nutrition 
of  the  eel]  and  the  other  definitely  associated  in  an  important  man- 
ner with  the  sexual  reproduction.  Multiplication  takes  place  by 
transverse  division  into  two  daughter  cells  or  by  budding.  In  the 
parasitic  forms  this  may  take  place  within  a  protecting  wall  (cyst). 


i6o 


GENERAL  BIOLOGY    OF   MICRO-ORGANISMS 


The  sexual  fertilization  is  not  followed  by  any  special  kind  of  divi- 
sion. Balantidium  is  the  only  genus  of  present  interest  as  a  cause 
of  human  disease.  See  Balantidium  coli  p.  435. 


OUTLINE  CLASSIFICATION  OF  MICRO-ORGANISMS. 


f  Phycomycetes 

Hyphomycetes 
(Molds) 

j  Basidiomycetes 
\  Ascomycetes 
|  Unclassified 

[    molds. 

[  Oidia 

Fungi  (Plants)    i 

Blastomycetes 
(Yeasts) 

J  Torulae 
1  Saccharomy- 

{    cetes. 

f  Trichobacteria 

Schizomycetes 

J  Coccaceae 

(Bacteria) 

j  Bacteriaceae 

Protista  (Micro- 

( Spirillaceae 

organisms) 

/  Spirochaetes 
Not  classified      \  ~,,      ,  ,       .      , 
I  Filterable  microbes 

f  Mastigophora 

f  Plasmodroma 

\  Rhizopoda 

Protozoa 

[  Sporozoa 

(Animals) 
j  Ciliophora 

f  Ciliata 
1  Suctoria. 

Specific  Nomenclature. — A  species  is  properly  designated  only 
by  a  binomial  Latin  name,  the  first  member  being  that  of  the 
genus,  such  for  example  as  Mucor  mucedo,  Saccharomyces  cere- 
visiae,  Bacillus  coli,  Spirochoeta  pallida,  Plasmodium  falciparum, 
and  Balantidium  coli.  A  third  term  may  be  employed  to  des- 
ignate a  variety  of  a  species,  but  such  usage  should  not  be  per- 
sisted in.  It  is  possible  to  give  the  variety  a  new  specific  name 


MORPHOLOGY   AND    CLASSIFICATION  l6l 

if  the  distinction  is  of  sufficient  importance,  or  to  drop  the  dis- 
tinctive term  from  the  Latin  name  altogether  if  the  difference 
proves  to  be  unimportant.  The  system  of  genera  is  in  a  very 
unsatisfactory  state,  especially  in  the  schizomycetes  where  the 
number  of  species  in  one  genus  is  much  too  large.  Even  in  the 
other  great  groups  the  generic  nomenclature  is  far  from  settled. 
The  specific  name  however  should  be  a  very  definite  and  single 
term,  and  it  is  usually  either  the  first  published  name  given  to 
the  organism  or  some  emended  adaptation  of  it,  in  proper 
grammatical  agreement  with  the  generic  term  employed.  Thus, 
in  designating  the  parasite  of  syphilis,  one  may  employ  the  term 
Spirochceta  pallida  classing  it  in  the  genus  Spiroch&ta  (Ehren- 
berg),  but  if  he  adopts  the  proposed  genus  Treponema  (Schau- 
dinn)  the  name  becomes  Treponema  pallidum. 


CHAPTER  VIII. 
PHYSIOLOGY    OF    MICRO-ORGANISMS. 

Relations  of  Morphology  and  Physiology. — In  morphological 
study  observations  are  restricted  to  the  relationship  of  various 
elements  at  a  given  time,  facts  relating  to  form  and  structure. 
From  the  physiological  viewpoint  one  is  more  interested  in  the 
sequence  of  events  and  the  relation  of  cause  and  effect.  The 
possible  suggestion  that  these  two  methods  of  study  are  independ- 
ent or  mutually  exclusive  would  be  most  unfortunate  and  is  really 
very  fallacious.  The  sequence  of  events  may  often  best  be  ascer- 
tained by  a  series  of  morphological  observations  of  a  microbe 
undergoing  change  of  form,  and  certainly  the  form  and  structure 
of  a  living  organism  at  a  given  time  may  be  properly  regarded  as 
an  expression  and  result  of  previous  physiological  activity  as  well 
the  most  essential  element  in  its  potentiality  for  future  activity. 
All  must  agree  that  difference  in  behavior,  that  is,  reaction  to  a 
definite  environmental  change,  is  really  associated  with  a  difference 
in  structure  of  the  living  organism.  The  important  difficulty 
lies  in  the  fact  that  the  morphological  or  structural  difference 
with  which  this  difference  in  reaction  is  correlated,  may  not  be 
capable  of  direct  observation  by  any  known  method  and  may  be 
ascertainable  only  by  means  of  the  physiological  test.  On  the 
other  hand  the  method  of  experimental  physiology  involves  the 
factor  of  environment,  small  and  unmeasured  differences  in  which 
may  grossly  influence  the  resulting  phenomenon  and  lead  to  erro- 
eous  conclusions.  Furthermore,  the  experimental  conditions  and 
the  method  of  physiological  observations  may  be  wholly  lacking 
in  adaptation  to  potentialities  of  the  organisms  under  observation. 
When  properly  employed,  however,  the  method  of  experimental 
physiology  yields  valuable  knowledge  obtainable  in  no  other  way, 

162 


PHYSIOLOGY   OF   MICRO-ORGANISMS  163 

and  it  has  been  the  most  important  single  method  in  establishing 
our  modern  ideas  of  the  relation  of  micro-organisms  to  infectious 
diseases,  and  is  the  method  of  greatest  promise  for  the  immediate 
futrue. 

Conditions  of  Physiological  Study. — The  physiology  of  many 
organisms  is  subject  to  only  very  limited  experimental  investi- 
gation. Those  organisms  of  very  narrow  biological  adaptation, 
such  as  many  of  the  parasitic  protozoa,  can  be  studied  only  in 
very  close  relation  to  their  natural  environment,  the  various 
important  elements  of  which  are  not  readily  subject  to  experi- 
mental alteration  and  are  largely  unrecognizable.  Our  knowl- 
edge of  these  forms  must  therefore  be  derived  almost  exclusively 
from  observations  of  form  and  structure,  physical  and  chemical, 
as  they  exist  and  change  under  the  natural  conditions  of  environ- 
ment, and  from  changes  which  take  place  in  the  tissues  surround- 
ing the  parasite,  which  we  may  ascribe  with  more  or  less  justifica- 
tion to  their  activity.  Practically  all  that  we  know  about  the 
physiological  activity  of  the  very  numerous  microbes  not  yet 
brought  into  the  group  of  artificially  cultivable  forms,  has  been 
deduced  from  morphological  observations.  Even  observations 
of  this  kind,  however,  can  be  more  successfully  pursued  in  those 
forms  capable  of  artificial  culture,  and  artificial  culture  is  a  prime 
necessity  for  the  study  of  cause  and  effect  by  the  methods  of  ex- 
perimental physiology.  For  this  reason  accurate  knowledge  of 
what  micro-organisms  do  is  much  richer  in  regard  to  the  culti- 
vable forms  such  as  bacteria,  yeasts  and  molds.  In  fact  the  mi- 
crobic  pure  culture  presents  the  most  favorable  object  known  for 
the  study  of  cellular  physiology  and  bio-chemistry.  Further- 
more, the  physiological  activities  of  many  microbes  are  of  the 
greatest  practical  importance.  It  is  not  surprising,  therefore, 
that,  among  the  bacteria,  many  of  which  grow  in  artificial  media 
under  a  great  variety  of  environmental  conditions,  the  relative 
ease  of  physiological  experimentation,  as  compared  with  the  dif- 
ficulty of  observation  of  the  minute  morphological  details,  and 
the  great  practical  importance  of  its  results  has  lead  to  an  enor- 


164  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

mo  us  development  of  knowledge  gained  by  the  first- mentioned 
method,  which  quite  over-shadows  our  knowledge  of  morph- 
ology and  structure  in  this  group  of  organisms. 

THE  INFLUENCE  OF  ENVIRONMENTAL  FACTORS. 

Moisture. — Moisture  is  indispensable  to  the  growth  of  mi- 
cro-organisms. A  few  species  will  grow  and  multiply  in  almost 
pure  distilled  water.  Drying  causes  the  death  of  the  majority  of 
the  vegetating  cells,  of  some  more  readily  than  others,  while  the 
spore  forms  may  remain  alive  in  a  dry  condition  for  many  years. 

Heim1  found  that  pathogenic  bacteria  resist  drying  much 
longer  when  contained  in  pathological  tissues  or  exudates  from 
animals  which  have  succumbed  to  the  disease,  than  when  they 
are  taken  from  artificial  cultures. 

Organic  food. — One  species  of  bacteria,  Nitrosomonas  of 
Winogradsky,  lives,  grows  and  multiplies  without  organic  food, 
utilizing  the  gases  of  the  atmosphere  as  its  source  of  carbon  and 
nitrogen.  From  the  standpoint  of  nutrition  this  organism  is 
among  the  most  primitive  of  beings.  Other  bacteria  are  known 
which  may  grow  in  water  containing  only  mineral  salts  and  a 
simple  sugar,  utilizing  large  quantities  of  atmospheric  nitrogen. 
These  are  known  as  nitrogen-fixing  bacteria.  Most  of  the  bac- 
teria, yeasts  and  molds  require  a  small  amount  of  nitrogenous 
organic  matter  as  food,  such  as  the  ammo-acids  or  albumoses,  and 
many  of  them  flourish  better  when  furnished  a  fermentable 
carbohydrate  such  as  dextrose.  The  complex  organic  molecules 
are  utilized  in  part  to  build  up  the  substance  of  the  bacteria,  but 
a  much  larger  part  of  them  is  broken  down  into  simpler  and  more 
stable  substances,  such  as  carbon  dioxide,  simple  fatty  acids, 
ammonia  and  water,  with  the  liberation  of  energy.  Sapro- 
phytic  organisms  are  those  which  grow  on  dead  organic  matter. 
Micro-organisms  of  still  narrower  adaptibility  grow  well  in  artifi- 
cial culture  only  if  they  be  furnished  abundant  protein  or  nucleo- 

1  Zeitschriftf.  Hygiene,  Apr.  4,  1905,  Bd.  L,  No.  i,  p.  123. 


PHYSIOLOGY    OF   MICRO-ORGANISMS  165 

protein.  Some  important  disease-producing  bacteria  belong  in 
this  category,  as  well  as  many  parasitic  spirochetes  and  some  _of 
the  protozoa.  Such  organisms  are  not  adapted  to  any  natural 
saprophytic  existence,  and  they  grow  in  the  artificial  cultures 
only  because  the  dead  medium  is  made  to  resemble  somewhat 
their  natural  parasitic  habitat.  Finally  there  are  the  micro-or- 
ganisms which  have  not  yet  been  grown  in  artificial  culture  and 
whose  food  requirements  are  essentially  unknown.  Many  of 
these  are  parasites,  and  are  called  obligate  parasites.  A  few  bac- 
teria, many  of  the  filterable  agents,  and  most  of  the  parasitic 
protozoa  are  included  in  this  category. 

Inorganic  Salts  and  Chemical  Reaction. — Phosphorus,  sul- 
phur, chlorine,  calcium,  sodium  and  potassium,  in  addition  to 
carbon,  hydrogen,  oxygen  and  nitrogen,  are  present  as  constituents 
of  the  microbic  protoplasm.  Minute  quantities  of  these  suffice  to 
supply  the  food  requirements  of  micro-organisms  and  it  is  un- 
necessary to  add  them  to  culture  media  to  serve  as  food.  Com- 
mon salt,  sodium  chloride,  is  ordinarily  employed  to  give  the 
artificial  medium  an  osmotic  tension  approaching  that  of  the 
body  fluids,  and  calcium  carbonate  is  sometimes  used  to  neutral- 
ize the  organic  acids  which  may  arise  in  the  culture  as  a  result  of 
the  bacterial  growth. 

The  most  favorable  chemical  reaction  for  most  micro-organisms 
is  that  of  actual  slight  alkalinity,  not  sufficiently  alkaline  to  pro- 
duce a  red  color  with  phenolphthalein  and  not  sufficiently  acid 
to  produce  a  red  color  with  litmus.  Some  bacteria  and  many  of 
the  yeasts  and  molds  will  grow  well  in  a  weakly  acid  medium, 
but  most  parasitic  bacteria  and  protozoa,  which  can  be  cultivated 
at  all,  require  a  reaction  slightly  alkaline  to  litmus  or  rosolic 
acid.  The  anaerobic  bacteria  do  best  in  a  medium  containing 
glucose  and  with  a  reaction  quite  alkaline,  indeed  very  close  to 
the  point  at  which  phenolphthalein  becomes  pink.  Organisms 
which  produce  acid  or  alkali  are  usually  arrested  in  their  growth  as 
soon  as  a  certain  concentration  is  reached,  and  the  medium  may 
then  rapidly  kill  the  micro-organisms. 


1 66  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

Oxygen. — Oxygen,  either  free  as  atmospheric  oxygen  or  com- 
bined as  in  water  or  organic  compounds,  is  an  essential  constitu- 
ent of  the  food  of  all  micro-organisms.  The  concentration  of 
uncombined  oxygen  dissolved  in  the  medium,  or  the  partial 
pressure  of  atmospheric  oxygen,  is  the  factor  ordinarily  meant 
when  oxygen  requirement  is  mentioned.  Many  micro-organisms 
grow  best  in  a  medium  freely  exposed  to  the  air.  These  are 
called  aerobes.  Some  which  will  grow  only  when  there  is  free 
access  of  oxygen  are  called  obligate  aerobes.  There  are  a  few 
bacteria  and  some  spirochetes  which  grow  only  in  the  absence 
of,  or  in  extremely  weak  concentration  of  oxygen.  These  are 
called  obligate  anaerobes.  Many  of  the  bacteria  grow  well  in 
various  concentrations  of  oxygen  or  in  its  absence.  These  are 
spoken  of  as  facultative  anaerobes,  or  sometimes  as  facultative 
aerobes  if  they  seem  to  prefer  the  anaerobic  existence.  Finally 
there  are  a  few  organisms,  some  bacteria  and  spirochetes,  and 
perhaps  some  protozoa,  which  seem  to  require  a  fairly  definite 
partial  pressure  of  oxygen,  but  are  not  adapted  to  growth  in 
a  medium  freely  exposed  to  the  atmosphere  (B.  bifidus,  B.  abortus, 
Spirochceta  rossii,  Plasmodium  falciparum) . 

Temperature. — Among  the  various  micro-organisms  are  found 
types  which  are  adapted  for  growth  at  different  temperatures 
throughout  a  considerable  range.  There  are  some  bacteria  and 
perhaps  some  molds  capable  of  growth  at  a  temperature  of  —0.5° 
C.,  in  food  substances  such  as  milk,  which  are  not  frozen  at  this 
temperature.  A  certain  yeast  is  said  to  multiply  even  at  —  6°  C., 
in  salted  butter.  Microbes  which  grow  at  very  high  temperatures, 
even  up  to  80°  C.,  occur  in  the  soil,  in  ensilage  and  sometimes 
in  the  intestine  of  animals.  The  great  majority  of  micro-organ- 
isms grow  only  between  o°  and  40°  C.  It  is  possible  to  recognize 
a  minimum,  a  maximum  and  an  intermediate  optimum  tem- 
perature for  growth  of  each  species.  Ordinarily  the  optimum 
temperature  is  only  a  few  degrees  below  the  maximum  at  which 
growth  will  take  place.  The^following  table  from  Marshall's 
Microbiology  illustrates  the  relation  of  these  temperatures. 


PHYSIOLOGY   OF   MICRO-ORGANISMS 


I67 


Temperatures 

opecies 

Minimum 

Optimum 

Maximum 

Penicillium  gJaucum            .    . 

I.c° 

250-27° 

3i°-36° 

As  per  gill  us  niger 

7°-io° 

77°-37° 

4o°-43° 

Saccharomyces  cerevisicz  I  

i°-  3° 

28°-3o° 

40° 

Saccharomyces  pasteurianus  I  
Bacterium  phosphoreum  

0-5° 
below  o° 

25°-30° 
i6°-i8° 

34° 
28° 

Bacillus  subtilis  

6° 

30° 

50° 

Bacterium  anthracis 

10° 

70°-37° 

43° 

Bacterium  ludwigii  

50° 

SS°-S7° 

80° 

Heating  above  the  maximum  temperature  for  growth  injures 
the  microbe  and  exposure  for  a  short  time  kills  it.  A  temperature 
of  60°  C.  for  20  to  30  minutes  destroys  most  vegetative  forms  of 
bacteria.  Cooling,  on  the  other  hand,  merely  checks  and  inhibits 
growth.  Freezing  destroys  some  of  the  germs  contained  in  a 
liquid  but  many  of  them  remain  alive.  Still  lower  temperatures 
seem  to  be  entirely  without  further  effect.  Bacteria  gradually 
die  in  frozen  material. 

Germicides. — Unfavorable  environmental  factors,  germicides 
and  antiseptics  have  been  considered  in  an  earlier  Chapter 
(Chapter  II). 

Microbic  Variation. — A  microbic  species  is  very  stable  in  its 
characters  when  maintained  under  fairly  constant  conditions  in 
its  normal  habitat.  Change  in  environment  brings  about  rather 
quickly  change  in  some  of  the  characters  of  a  bacterial  species. 
The  alterations  in  virulence  or  ability  to  produce  disease,  which 
may  be  produced  by  methods  of  artificial  culture,  are  perhaps 
best  known.  It  would  seem  that  the  descendants  of  a  single 
cell  are  not  all  identical,  but  they  vary  among  themselves  within 
fairly  narrow  limits  in  respect  to  a  great  many  characters,  fluc- 
tuating about  a  mean  type  which  is  that  best  adapted  to  the 
environment.  With  a  change  in  surrounding  conditions,  this 
mean  or  normal  type  may  no  longer  be  best  adapted,  but  a 


1 68  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

variation  slightly  removed  in  respect  to  certain  characters  may 
flourish  better  and  become  the  mean  type  about  which  the 
fluctuating  variants  group  themselves.  Thus  the  pure  culture 
seems  to  respond  to  environmental  change.  Whether  the 
fluctuating  variations  are  due  to  small  differences  in  the  imme- 
diate surroundings  of  the  individual  microbes,  or  whether  they 
arise  as  a  result  of  a  property  of  variability  inherent  in  protoplasm, 
may  be  disputed,  but  the  latter  view  is  more  commonly  held  by 
biologists. 

THE  PRODUCTS  OF  MICROBIC  GROWTH. 

The  effects  resulting  from  the  growth  of  a  micro-organism 
depend  on  the  one  hand  upon  the  nature  of  the  organism  and  on 
the  other  upon  the  environment,  more  especially  the  medium  in 
which  it  grows  and  the  conditions  of  temperature  and  oxygen 
supply.  Apparently  slight  variations  in  the  latter  may  influence 
the  results  to  a  marked  degree. 

Physical  Effects. — Heat  is  evolved  by  many  actively  growing 
bacterial  cultures  and  is  especially  evident  in  the  fermentation 
of  such  substances  as  ensilage  and  manure.  Perhaps  some  of 
the  heat  may  result  directly  from  microbic  activity,  but  the  most 
of  it  appears  to  arise  from  secondary  chemical  reactions  in  which 
the  microbic  products  sometimes  play  a  part.  Microbes  which 
produce  heat  are  designated  as  thermogenic.  Light  is  also  emitted 
by  some  microbic  cultures.  Here  it  seems  certain  that  the  light 
is  produced  by  the  oxidation  of  a  bacterial  product  and  not  emitted 
directly  by  the  micro-organisms.  These  phosphorescent  or 
photogenic  organisms  occur  in  salt  water  and  on  fish  and  they 
have  rarely  been  found  in  other  places. 

Chemical  Effects. — These  are  the  most  important  results  of 
microbic  growth.  As  we  have  just  seen,  the  production  cf 
heat  and  light  is  probably  due  to  a  secondary  reaction  entered 
into  by  some  of  the  chemical  products  of  growth.  Almost  all 
the  other  important  practical  effects  of  the  growth  of  micro- 


PHYSIOLOGY   OF   MICRO-ORGANISMS  169 

organisms  are  due  to  chemical  changes  produced  by  them. 
Primary  products  are  those  which  are  produced  inside  the  cell 
by  its  living  protoplasm.  These  include  all  the  synthetic  products 
such  as  the  substance  of  the  germ  itself,  the  complex  bodies 
which  it  forms  from  simpler  substances,  such  as  its  enzymes 
and  its  toxins,  and  also  the  simpler  chemical  substances  which 
result  from  internal  cellular  metabolism,  the  proper  excretions 
of  the  cell.  The  secondary  products  are  those  which  result  from 
the  action  of  a  primary  product,  such  as  an  enzyme,  upon  some 
material  outside  the  cell.  The  distinction  is  clear  enough  in 
theory  but  practically  it  is  often  obscure. 

Enzymes. — Fermentation  in  its  broad  sense  means  the 
chemical  changes  brought  about  by  living  cells  or  their  products. 
In  its  more  restricted  sense,  it  applies  to  the  splitting  of  carbohy- 
drates by  the  action  of  microbes,  which  is  accompanied  by  the 
evolution  of  gas.  Organisms  which  cause  active  fermentation 
are  spoken  of  as  zymogenic.  Dextrose,  C6Hi2O6,  is  a  readily 
fermentable  carbohydrate  and  is  decomposed  in  various  ways 
by  different  microbes.  In  some  instances  a  large  proportion 
of  it  is  converted  into  alcohol  and  carbon  dioxide  according  to 
the  following  equation: 

C6Hi2O6(fermented)  =  2C2H6O+2CO2. 

Other  kinds  of  micro-organisms  produce  little  alcohol  or  gas 
but  abundant  lactic  acid.  The  reaction  may  be  represented 
roughly  by  this  equation: 

C6Hi206(fermented)  =  2C3H6O3. 

In  other  instances  acetic  acid  may  be  produced: 

C6Hi2O6(fermented)  =3C2H4O2. 

These  equations  are  only  an  approximate  indication  of  the 
reactions  which  take  place,  as  it  is  very  doubtful  that  the  whole 
molecule  of  dextrose  is  ever  converted  into  a  single  simpler 
compound  by  fermentation,  but  they  will  serve  to  indicate  the 


170  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

nature  of  the  reactions  involved  and  to  suggest  the  variety  of 
products  which  may  arise  from  the  decomposition  of  complex 
organic  substances.  Some  of  these  fermentative  changes  take 
place  to  a  large  extent  inside  the  microbic  cell.  Such  is  the 
case  in  the  alcoholic  fermentation  produced  by  saccharomyces. 
The  sugar-splitting  or  glycolytic  ferments  are  found  in  the 
cultures  of  many  bacteria  and  molds.  Less  common  are  the 
diastatic  ferments  capable  of  changing  starch  to  dextrose,  the 
inverting  ferments  which  change  saccharose  and  lactose  into 
glucose  and  other  hexoses,  and  the  acetic  ferments  capable  of 
causing  the  oxidation  of  alcohol  to  produce  vinegar. 

The  fermentation  or  decomposition  of  proteins  usually  gives 
rise  to  evil-swelling  gases.  This  decomposition  is  called  putrefac- 
tion, and  the  organisms  which  cause  it  are  called  saprogenic  or 
putrefactive  organisms.  The  nature  of  the  products  is  much 
influenced  by  the  amount  of  oxygen  available  and  the  foulest 
gases  are  produced  especially  in  the  absence  of  oxygen.  Proteo- 
lytic  ferments  of  the  same  general  nature  as  trypsin  are  produced 
by  many  microbes.  A  few  form  rennet-like  enzymes.  Proteo- 
lytic  ferments  which  act  in  the  presence  of  acid,  like  pepsin,  are 
produced  by  some  molds  and  by  some  bacterial  species. 

The  decomposition  of  the  complex  protein  molecules  gives  rise 
to  an  enormous  variety  of  intermediate  products  before  the  ulti- 
mate analysis  into  ammonia,  carbon  dioxide,  water,  sulphates  and 
phosphates  is  accomplished.  Many  of  these  intermediate  prod- 
ucts are  very  unstable  and  of  unknown  chemical  composition. 
Some  of  them  are  highly  poisonous.  Brieger  and  his  followers 
were  able  to  separate  a  number  of  the  complex  substituted  ammo- 
nia and  ammonium  compounds  in  a  pure  state  and  these  par- 
ticular bodies  are  known  as  putrefactive  alkaloids,  or  as  ptomains. 
A  simple  ptomain  is  trimethylamin,  N(CH3)3;  a  more  complex 
one  cadaverin,  HgN-CHa'CHa-CHa'CHa'CHa-NHa.  Some  of  the 
ptomains  are  poisonous.  These  various  decomposition  products 
are  for  the  most  part  secondary  products  resulting  from  the  action 
of  enzymes  upon  the  decomposing  material.  Many  of  them  are 


PHYSIOLOGY   OF   MICRO-ORGANISMS  171 

so  unstable  that  their  presence  in  a  decomposing  substance  is 
influenced  by  access  of  air,  temperature  and  moisture,  and  they 
may  quickly  disappear  or  decompose. 

Micro-organisms  also  form  fat-splitting  or  steatolytic  enzymes, 
and  enzymes  capable  of  transforming  urea  into  ammonium 
carbonate. 

NH2-CO-NH2+2H2O  (fermentation)  =  (NH4)2CO3. 

Various  inorganic  substances  undergo  chemical  change  under  the 
influence  of  microbic  activity  and  some  of  these  changes  appear  to 
be  due  to  enzymes.  Specific  examples  will  be  considered  in  the 
section  on  the  soil  bacteria. 

The  toxins  of  bacteria  are  primary  products  built  up  by  the  cell. 
The  true  bacterial  toxins  are  of  unknown  chemical  composition, 
are  labile  like  enzymes  and  stimulate  the  production  of  antitoxins 
when  they  are  injected  into  animals.  They  are  the  most  poisonous 
substances  at  present  known.  Analogous  substances  have  been 
found  in  some  plants,  ricin  in  the  castor  bean  and  abrin  in  the 
jequirity  bean,  and  the  poisonous  property  of  some  kinds  of  snake 
venom  is  due  to  the  presence  of  substances  similar  in  nature  to 
the  bacterial  toxins.  These  substances  will  be  considered  more 
fully  in  a  later  chapter  devoted  to  the  relation  of  parasitic  microbes 
to  their  hosts. 

MUTUAL  RELATIONS  OF  A  MICROBE  AND  ITS  ENVIRONMENT. 

Morphological  Characters. — It  is  evident  that  the  phenomena 
of  growth  taking  place  in  a  microbic  pure  culture  depend  not  only 
upon  the  particular  kind  of  microbe  present  but  also  in  a  very 
important  way  upon  the  chemical  and  physical  structure  of  the 
medium,  the  access  of  air  and  the  temperature.  Variations  in 
these  latter  may  even  bring  about  considerable  alteration  in  the 
form  and  structure  of  the  individual  cells.  A  common  effect  of 
high  temperature  is  the  shortening  of  individual  bacilli  and  spirilla 
because  of  more  rapid  division  and  complete  separation  of  the 


172  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

daughter  cells.  The  presence  of  unfavorable  influences,  such  as 
antiseptics  or  bacterial  waste  products  in  the  medium,  may  cause 
marked  irregularities  in  the  shape  and  size  of  the  cells,  so-called 
involution  forms.  The  ability  to  form  endospores  may  be  lost 
through  growth  at  high  temperature.  The  form  which  a  micro- 
organisms presents  in  a  given  instance  may  not,  therefore,  be 
regarded  as  essentially  typical  without  regard  to  the  conditions 
under  which  it  has  been  produced. 

The  morphology  of  cell-groups  is  even  more  obviously  depend- 
ent upon  the  conditions  of  the  environment  and  the  physiological 
properties  of  the  micro-organism.  A  slow  scanty  growth  on  a 
given  medium  does  not  necessarily  mean  that  the  organism 
essentially  lacks  vigor.  It  may  mean  that  the  medium  is  not  well 
adapted  to  the  requirements.  Diffuse  growth  through  a  semi- 
solid  medium  may  be  merely  an  expression  of  the  motility  of  an 
organism.  A  great  variety  of  different  culture  media  have  been 
employed  to  bring  out  more  or  less  characteristic  features  in  the 
gross  appearance  of  cultures,  and  these  appearances  often  depend 
upon  the  grouping  of  the  cells  or  upon  their  fermentative  activity 
or  both.  Although  the  characters  of  a  cell-group  of  micro-organ- 
isms are  really  morphological  characters  of  the  same  general  na- 
ture as  the  morphological  characters  of  higher  plants  and  animals, 
to  which  so  much  significance  is  attached,  in  the  case  of  micro- 
organisms in  an  artificial  environment,  such  as  a  culture  medium, 
the  gross  appearance  or  the  cell-grouping  is  more  properly  regarded 
as  a  feature  of  physiological  rather  than  morphological  significance. 
Nutrient  gelatin  is  a  medium  well  adapted,  in  the  case  of  those  mi- 
crobes which  will  grow  in  it,  for  showing  physiological  differences 
in  the  appearance  of  cell-groups  or  colonies,  and  perhaps  a  greater 
variety  of  appearances  may  be  obtained  upon  this  medium  than 
any  other.  Unfortunately  its  use  entails  certain  difficulties,  the 
most  important  of  which  is  the  necessity  for  experience  andscare 
in  the  interpretation  of  the  appearances  observed.  Important 
features  in  the  appearance  of  the  colonies  and  other  cell-groups 
are  brought  out  by  the  use  of  various  other  media. 


PHYSIOLOGY   OF   MICRO-ORGANISMS  173 

Physiological  Tests. — Specific  tests  for  a  simple  physiological 
character  require  less  skill  and  care  in  their  observation,  and  are 
widely  used.  Cultivation  in  a  fermentation  tube  of  sugar  broth 
as  a  test  of  ability  to  form  gas  from  the  sugar,  titration  of  sugar- 
broth  cultures  to  ascertain  the  ability  to  produce  acid  from  various 
sugars,  chemical  test  for  the  presence  of  indol  and  of  ammonia  in  a 
culture  in  peptone  solution,  observation  of  the  ability  to  hemolyze 
or  discolor  blood  mixed  with  the  medium,  and  the  ability  to  fer- 
ment glycerin,  these  are  some  of  the  valuable  simpler  tests. 
Cultivation  in  milk  is  a  somewhat  more  complex  test,  as  a  variety 
of  fermentable  substances  is  offered  the  microbe,  increasing  the 
difficulties  of  interpretation  but  also  increasing  the  variety  of  phe- 
nomena which  may  occur. 

A  convenient  outline  to  use  in  making  morphological  and 
physiological  observations  upon  bacteria  and  in  recording  the  re- 
sults, has  been  prepared  by  a  committee  of  the  Society  of  American 
Bacteriologists.  Many  features  of  this  will  be  found  of  assistance 
in  the  study  of  new  or  unknown  bacteria,  especially  saprophytic 
forms.  A  copy  of  the  revised  descriptive  chart  is  inserted. 


CHAPTER  IX. 

THE  DISTRIBUTION  OF  MICRO-ORGANISMS  AND 
THEIR  RELATION  TO  SPECIAL  HABITATS. 

General  Distribution. — Micro-organisms  are  very  generally 
distributed  over  the  surface  of  the  earth  and  in  its  waters,  and 
are  carried  about  as  dust  in  the  air.  They  flourish  abundantly 
in  the  digestive  canals  of  animals  and  on  their  body  surfaces. 
Wherever  there  is  organic  matter,  the  dead  remains  of  animal 
and  plant  life,  there  are  micro-organisms  in  abundance  living 
upon  the  dead  material  and,  if  the  temperature  and  moisture  be 
suitable,  transforming  it  into  simpler  chemical  substances.  In 
the  soil,  bacteria,  yeasts,  molds  and  protozoa  are  fairly  numerous, 
especially  in  fertile  soils  near  the  surface.  Their  number  rapidly 
diminishes  in  the  deeper  layers,  and  at  a  depth  of  six  to  twelve 
feet  they  are  very  scarce  or  entirely  absent.  The  surface  waters 
of  the  earth  contain  large  numbers  of  bacteria  and  protozoa, 
especially  numerous  where  organic  matter  is  abundant.  The  air 
contains  considerable  numbers  of  molds  and  bacteria  suspended 
as  dust.  The  deep  layers  of  the  soil  and  water  below  impervious 
rock  strata  are  free  from  micro-organisms.  The  surfaces  of  snow- 
covered  mountains  and  of  the  frozen  polar  regions  of  the  earth,  as 
well  as  the  atmosphere  in  these  regions,  are  practically  free  from 
microbes.  The  atmosphere  over  large  bodies  of  water  during 
calm  weather,  the  air  in  damp  cellars,  in  sewers  and  in  undisturbed 
rooms  is  germ-free,  because  the  suspended  dust  particles  settle 
out  and  do  not  escape  again  into  the  air  unless  swept  up  by  air 
currents,  which  must  be  rather  violent  to  remove  them  from 
moist  surfaces. 

174 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  175 

The  environment  and  the  surfaces  of  growing  plants  and  animals 
are  rich  in  micro-organisms,  especially  bacteria,  but  the  interior 
of  the  living  tissues  is  generally  germ-free  in  health.  To  this 
statement  there  are  certain  exceptions,  namely,  the  occurrence 
of  a  few  bacteria  in  the  liver,  the  thoracic  duct  and  the  blood  of 
animals  during  active  digestion,  which  are,  however,  soon  de- 
stroyed by  the  healthy  tissue;  and  the  invasion  of  the  root  tissues 
of  leguminous  plants  byPs.radicicola  and  the  growth  of  the  bac- 
terium within  the  plant  tissues,  which  results  not  in  injury  to 
the  host  but  in  a  definite  improvement  of  its  nutrition  by  enabling 
it  to  utilize  atmospheric  nitrogen. 

Micro-organisms  of  the  Soil. — The  germ-content  of  soil  depends 
chiefly  upon  the  amount  of  organic  matter  present.  They  may 
be  present  in  millions  per  gram  of  soil.  Bacteria,  molds  and  pro- 
tozoa are  the  most  numerous.  Their  relation  to  soil  fertility 
seem  to  be  important,  and  they  probably  play  a  large  part  in 
preparing  the  organic  matter  of  the  soil  for  use  as  food  by  plants. 
A  great  many  soil  bacteria  decompose  protein  and  set  free  am- 
monia, and  the  urea  bacteria  are  especially  important  in  the 
transformation  of  urea  and  of  animal  manures  into  ammoniacal 
compounds.  The  transformation  of  ammoniacal  compounds 
into  nitrates,  so-called  nitrification,  is  accomplished  by  the  nitri- 
fying bacteria,  of  which  a  few  species  have  been  obtained  in  pure 
culture,  Nitrosomonas  of  Winogradski  which  produces  nitrite 
from  ammonia,  and  his  genus  Nitrobacter  which  oxidizes  nitrites 
to  nitrates.  Very  many  species  of  soil  bacteria  are  able  to  change 
nitrogen  in  the  opposite  direction,  reducing  nitrates  to  nitrites 
and  further  to  ammonia  or  to  free  nitrogen  gas.  Of  special  interest 
are  the  soil  bacteria  which  are  able  to  fix  atmospheric  nitrogen, 
that  is,  absorb  nitrogen  from  the  air  and  combine  it  so  as  to  make 
it  available  for  plant  food.  The  various  species  of  the  genus 
Azotobacter  (A.  chroococcum,  A.  beyerincki)  accomplish  this  as 
they  grow  in  the  presence  of  dextrose,  and  the  organism  of  the 
root  tubercles,  Pseudomonas  radicicola,  fixes  nitrogen  as  it  grows 
within  the  tissues  of  the  legume  roots.  Numerous  soil  bacteria 


176  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

ferment  sugars,  starches  and  fats,  and  there  are  several  known 
species  capable  of  dissolving  cellulose.1 

Pathogenic  Soil  Bacteria. — Certain  pathogenic  bacteria  are 
of  common  occurrence  in  the  soil.  Whether  this  is  their  normal 
habitat  or  whether  they  gain  entrance  to  the  soil  with  animal 
excrement,  may  be  questioned.  At  any  rate  the  pathogenic 
anaerobes,  B.  edematis,  B.  tetani,  and  B.  welchii  are  likely  to  occur 
in  garden  soil,  and  it  seems  probable  that  they  actually  multiply 
there  to  some  extent.  Bact.  anthracis  also  occurs  in  the  soil  of 
fields  where  the  disease  has  prevailed,  and  it  is  not  improbable 
that  this  organism  multiplies  in  the  ground  at  times.  Other 
pathogenic  bacteria,  such  as  those  of  typhoid  and  cholera,  seem 
to  be  rather  quickly  eliminated  in  the  struggle  for  existence  under 
the  conditions  found  in  surface  soils. 

Micro-organisms  of  the  air. — Micro-organisms  exist  in  the  air 
only  as  floating  particles  of  dust,  or  as  passengers  on  small  drop- 
lets of  moist  spray,  or  as  parasites  on  or  in  winged  aerial  creatures. 
Those  floating  as. dust  are  derived  from  the  earth's  surface,  and 
most  of  the  living  germs  usually  found  in  this  condition  are  the 
spores  of  molds.  Living  tubercle  bacilli  are  unquestionably 
suspended  in  the  air  as  dust,  especially  in  the  dry  sweeping  of 
floors  where  careless  consumptives  have  lived.  The  spores  of 
anthrax  bacilli  may  also  be  suspended  in  the  air  where  hides  or 
wool  of  anthrax  animals  are  handled.  Other  pathogenic  bacteria 
may  at  times  float  as  dust,  but  their  presence  in  the  air  in  this 
condition  is  apparently  rather  uncommon,  and  should  be  expected 
only  in  the  fairly  recent  environment  of  cases  of  the  disease.  The 
moist  droplets,  expelled  from  the  mouth  and  nose  in  speaking, 
in  coughing  and  especially  in  sneezing,  may  remain  suspended  in 
the  air  for  many  minutes  and  be  distributed  to  considerable 
distances.  After  drying  the  solid  material  may  still  float  as  dust. 
Pathogenic  micro-organisms  may  readily  be  transmitted  from 
person  to  person  in  this  way. 

1  For  a  discussion  of  the  microbiology  of  the  soil,  see  Monograph  by  Lipman  in 
Marshall's  Microbiology,  1911. 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  177 

In  a  rough  way  one  may  obtain  some  knowledge  of  the  charac- 
ter of  the  organisms  in  the  air  of  a  given  locality  by  removing  the 
cover  of  a  Petri  dish  containing  sterilized  gelatin  or  agar  for  a 
few  minutes,  replacing  it,  and  allowing  the  organisms  to  develop. 
In  most  cases  a  large  proportion  of  the  growths  that  appear  will 
be  molds.  Yeasts  are  also  common,  and  among  the  bacteria 
the  micrococci  are  abundant.  Chromogenic  varieties  are  likely 
to  be  present. 

A  few  studies  of  this  character  will  show  that  the  number 
of  organisms  that  are  present  depends  chiefly  upon  whether  the 
air  is  quiet  or  has  recently  been  disturbed  by  drafts,  gusts  of 
wind,  or  sweeping.  These  facts  are  of  fundamental  importance 
in  laboratory  work,  if  we  wish  to  avoid  contaminations.  Among 
various  devices  that  have  been  proposed  for  the  accurate  study 


FIG.  82. — Sedgwick-Tucker  aerobioscope. 

of  the  organisms  of  the  air,  the  Sedgwick-Tucker  aerobioscope 
is  the  simplest  and  most  accurate.  It  consists  of  a  glass  tube, 
one  end  of  which  is  drawn  out  so  as  to  be  smaller  than  the  other. 
The  small  end  contains  a  quantity  of  fine  granulated  sugar; 
both  ends  are  plugged  with  cotton,  and  the  instrument  is  sterilized. 
A  definite  quantity  of  air  is  to  be  aspirated  through  the  large 
end,  after  removing  the  cotton,  and  this  may  be  done  by  means 
of  a  suction-pump  applied  to  the  other  end,  or  by  siphoning 
water  out  of  a  bottle,  the  upper  part  of  which  is  connected  with 
the  end  of  the  aerobioscope  by  means  of  a  rubber  tube.  The 
sugar  acts  as  a  filter  and  sifts  out  of  the  air  the  micro-organisms 
which  are  contained  in  it.  Liquefied  gelatin  or  agar  may  be 
introduced  into  the  large  end  of  the  instrument  by  means  of  a 
bent  funnel;  and,  after  replacing  the  cotton,  it  is  mixed  with 
the  sugar  which  dissolves.  The  culture-medium  may  be  spread 


178  GENERAL  BIOLOGY   OF  MICRO-ORGANISMS 

around  the  inside  of  the  larger  portion  of  the  tube  after  the 
manner  of  an  Esmarch  roll-tube.  The  bacteria  which  are  filtered 
out  by  the  sugar  will  develop  as  so  many  colonies  upon  the 
solidified  medium. 

Many  important  micro-organisms,  and  certainly  some  germs 
of  disease,  are  borne  through  the  air  by  the  winged  insects,  and 
to  a  less  extent  by  birds.  The  microbes  are  found  not  only  on 
the  feet  and  outer  body  surfaces  of  these  carriers,  but  they  also 
occur  on  and  in  the  mouth  parts,  in  the  alimentary  canal  and 
sometimes  in  the  interior  of  the  animal's  body  tissues.  Certain 
pathogenic  micro-organisms  (Plasmodium,  Trypanosoma)  are 
known  to  be  transmitted  from  one  person  to  another  almost 
exclusively  by  biting  insects,  and  the  importance  of  these  carriers 
in  air-borne  disease  of  both  animals  and  plants,  is  being  recognized 
more  and  more. 

Micro-organisms  of  Water  and  Ice. — The  water  of  rivers, 
lakes  and  the  ocean  always  contains  bacteria.  The  number 
of  organisms  varies  greatly  in  different  places  and  under  different 
conditions.  The  number  of  different  species  found  in  water 
is  also  very  large.  Some  of  these,  the  natural  water  bacteria, 
including  many  bacilli  which  produce  pigment  and  some  cocci 
and  spirilla,  seem  to  live  in  surface  water  as  their  natural  habitat. 
With  the  addition  of  putrescible  material  these  forms  are  in- 
creased in  number  and  certain  of  them  (Proteus  group,  fluorescing 
bacteria)  become  numerous.  Soil  bacteria  are  numerous  in 
waters  during  floods  and  after  rain,  and  they  may  persist  for 
some  time.  Intestinal  bacteria  occur  in  waters  which  receive 
sewage  or  are  otherwise  contaminated  with  excreta.  They 
persist  only  a  relatively  short  time.  Certain  intestinal  protozoa, 
•Entamaba,  Aalantidtum,  seem  also  to  occur  in  waters  at  times. 
Ground-water1  contains  few  or  no  bacteria  under  normal  condi- 
tions, and  is  therefore  suitable  for  a  source  of  water-supply, 
when  a  sufficient  amount  is  available.  The  possibility  of  contami- 

1  Ground-water  is  the  water  which — originally  derived  from  rain  or  snow — sinks 
through  superficial  porous  strata,  like  gravel,  and  collects  on  some  underlying, 
mpervious  bed  of  clay  or  rock. 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  179 

nation  of  the  ground-water  from  unusual  or  abnormal  conditions 
should  always  be  eliminated  before  it  is  taken  for  drinking 
water.  Numerous  epidemics  of  typhoid  fever  have  been  traced 
to  contamination  of  wells.  The  location  of  wells  with  reference 
to  privy-vaults  and  other  possible  sources  of  contamination 
should  be  chosen  with  the  greatest  care. 

The  ordinary  bacteria  of  water  are  harmless,  as  far  as  is 
known.1  Bad  odors  and  tastes  in  drinking  water  that  is  not 
polluted  with  putrid  material  are  usually  due  to  minute  green 
plants  (algae).2  The  diseases  most  commonly  disseminated 
by  water  are  typhoid  fever  and  Asiatic  cholera,  and  probably 
also  dysentery.  The  spirillum  of  cholera  will  usually  die  in 
natural  water  (not  sterilized  water)  inside  of  two  or  three  weeks;  the 
bacillus  of  typhoid  fever  will  usually  die  in  two  or  three  weeks. 
Under  exceptional  circumstances  these  organisms  may  perhaps 
maintain  their  vitality  for  a  longer  period.  They  appear,  however, 
to  be  less  hardy  than  the  ordinary  water  bacteria.  As 
we  now  understand  these  diseases,  the  organisms  causing  them 
will  be  present  only  in  a  water-supply  which  has  been  recently 
contaminated  by  the  excreta  from  a  case  of  the  disease.  Notwith- 
standing the  rapid  death  of  these  organisms  in  water,  they 
may  exist  long  enough  to  infect  individuals  habitually  drinking 
the  water.  Many  epidemics  of  cholera  and  typhoid  fever  have 
been  traced  to  water  polluted  with  the  discharges  from  cases 
of  these  diseases,  and  in  a  few  instances  the  relation  of  the  con- 
taminated water  supply  to  the  epidemic  has  been  established 
beyond  a  reasonable  doubt. 

By  self -purification  of  water  is  meant  the  removal,  through 
natural  processes,  of  contaminating  organisms  such  as  might 
occur  from  the  discharge  of  sewage  into  it.  It  depends  upon 
the  sedimentation  of  the  contaminating  material  in  the  form 
of  mud,  upon  the  growth  of  the  ordinary  water-plants  and  protozoa, 

1  See  Fuller  and  Johnson,  "The  Classification  of  Water  Bacteria,"  Journal  of 
Experimental  Medicine,  Vol.  IV,  p.  609. 

: "  Contamination  of  Water  Supplies  by  Algae."  G.  T.  Moore  in  Yearbook 
U.  S.  Department  of  Agriculture,  1902. 


180  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

upon  the  exhaustion  of  the  food  supply  by  the  growth  of  bacteria 
themselves,  upon  the  destructive  influence  of  direct  sunlight, 
and  the  dilution  of  the  contamination  by  a  large  volume  of 
water.1  It  is  not  usually  to  be  relied  upon  as  a  means  of  freeing 
the  water-supply  from  pathogenic  bacteria. 

Storage  of  Water. — When  water  is  kept  in  large  reservoirs, 
the  solid  particles  in  it,  including  bacteria,  tend  to  fall  to  the 
bottom.  The  number  of  bacteria  in  a  water-supply  may  be 
considerably  reduced  .in  this  way.  The  use  of  large  storage 
reservoirs  also  provides  for  the  dilution  of  any  sudden  excess  of 
pollution,  and  if  the  water  is  held  in  storage  the  pathogenic 
germs  present  disappear  for  the  most  part  in  a  few  days  or  weeks. 

Filtration. — Water  may  be  completely  sterilized  by  passing 
it  through  the  Pasteur-Chamberland  niters  of  unglazed  porcelain, 
or  through  the  more  porous  Berkefeld  niters.  Such  niters  are 
effective  only  when  frequently  cleaned  and  baked,  and  in  practical 
purification  of  water  for  household  purposes  they  usually  fail 
because  of  the  intelligent  care  they  require.  Other  types  of 
domestic  filters  are  generally  worse  than  useless. 

Filtration  on  a  large  scale  has  been  more  commonly  in  use  in 
the  cities  of  Europe  than  elsewhere,  until  lately.  Filtration- 
plants  now  exist  in  several  cities  of  the  United  States.  By  this 
method  98  per  cent  to  99  per  cent  of  the  bacteria  in  water  may 
be  removed. 

Slow  Sand  Filtration.2 — The  filter  consists  of  successive  layers 
of  stones,  coarse  and  fine  gravel.  The  uppermost  layers  are 
of  fine  sand.  The  whole  filter  is  from  i  to  2  meters  thick.  The 
sand  should  be  60  cm.  to  1.2  meters  in  thickness.  The  accumu- 
lated deposit  from  the  water  and  a  little  of  the  fine  sand  must 
be  removed  from  time  to  time,  but  the  layer  of  fine  sand  must 
never  be  allowed  to  become  less  than  30  cm.  in  thickness.  The 
first  water  coming  from  the  filter  is  discarded.  The  actual  fil- 
tration is  done  largely  by  the  slimy  sediment  which  collects 

1  See  Jordan,     Journal  of  Experimental  Medicine.     Vol.  V,  p.  271. 

2  For  a  full  discussion  see  Journal  American   Medical  Association.     Oct.  3  to 
3i,  1903- 


THE   DISTRIBUTION    OF   MICRO-ORGANISMS  l8l 

on  the  surface  of  the  layer  of  fine  sand.  The  filterbeds  may 
be  several  acres  in  extent,  and  in  cold  climates  should  be  pro- 
tected by  arches  of  brick  or  stone.  They  require  renewal  occa- 
sionally. This  kind  of  filtration  has  come  largely  into  use  since 
the  cholera  epidemic  of  1892-93,  and  it  appears  to  be  very  effective. 
It  is  important  to  use  storage  basins  in  connection  with  sand 
filtration. 

The  results  obtained  by  filtration  depend  greatly  upon  the 
intelligence  displayed  in  operation,  and  must  be  controlled  by 
frequent  examinations  of  the  water. 

Mechanical  Filtration. — This  method  of  filtration  is  also 
called  the  American  system.  It  is  more  rapid  than  the  preceding 
method  and  does  not  require  a  large  area  for  filter  beds.  Al- 
though sand  is  required  also,  filtration  is  accomplished  by  a 
jelly-like  layer  of  aluminium  hydroxide.  This  product  is  formed 
by  adding  to  the  water  a  small  quantity  of  aluminium  sulphate 
or  of  alum.  The  carbonates  in  the  water  decompose  the  aluminium 
salt  and  produce  aluminium  hydroxide.  It  precipitates  as  a 
white,  flocculent  deposit,  entangling  solid  particles,  including 
bacteria,  as  coffee  is  cleared  with  white  of  egg.  Only  a  'trace 
of  aluminium  should  appear  in  the  water.  This  method  of  filtra- 
tion has  not  been  tested  so  extensively  as  slow  sand  filtration, 
but  seems  likely  to  prove  efficient.  With  water  poor  in  carbonates, 
these  may  have  to  be  added.1 

Whipple  and  Longley2  found  that  the  efficacy  of  mechanical 
filters  with  the  addition  of  alum  depends  somewhat  upon  the 
character  of  the  alum.  They  find  that  the  alum  shall  be  shown 
by  analysis  to  contain  17  per  cent  of  alumina  (AljOj)  soluble 
in  water,  and  of  this  amount  at  least  5  per  cent  shall  be  in  excess 
of  the  amount  necessary  theoretically  to  combine  with  the 
sulphuric  acid  present.  It  shall  not  contain  more  than  i  per 
cent  of  i  soluble  substances,  and  shall  be  free  from  extraneous 
debris  of  all  kinds.  It  must  not  contain  more  than  0.5  per 

1  See  Fuller,  Journal  American  Medical  Association,  Oct.  31,  1903. 

2  Jonrn.  Infect.  Diseases,  Supplement  No.  2,  Feb.,  1906,  pp.  166—171. 


182  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

cent  of  iron  (Fe2O3)  and  the  iron  shall  be  preferably  in  the 
ferrous  state. 

Chemical  Disinfection. — Various  methods  for  the  purification 
of  water  by  means  of  chemicals  have  been  proposed.  The  use 
of  copper  sulphate  to  disinfect  drinking  water  was  recommended 
by  Moore  and  Kellerman,1  and  various  investigators  tested  the 
value  of  their  recommendation.  Clark  and  Gage2  came  to  the 
conclusion  from  their  investigation  that  the  treatment  of  water 
with  copper  sulphate  or  the  storing  of  water  in  copper  vessels 
has  little  practical  value.  Others  also  have  come  to  practically 
the  same  conclusion.  While  the  addition  of  copper  sulphate  is 
of  use  in  preventing  the  growth  of  the  algae,  which  sometimes 
grow  so  abundantly  as  to  choke  up  water  pipes,  and  is  of  benefit 
in  this  direction,  the  weight  of  evidence  appears  to  be  against 
its  efficacy  for  purifying  water  for  drinking  purposes.  More 
effective  chemical  disinfection  has  been  obtained  by  means  of 
ozone  generated  by  electricity.  More  recently,  calcium  hy- 
pochlorite  and  free  chlorine  have  been  employed  for  this  purpose 
with  considerable  success. 

Physical  Disinfection. — The  most  effective  and  surest  method 
of  disinfecting  drinking  water  is  by  boiling  it  or  by  distillation. 

Bacteriological  Examination  of  Water. — For  bacteriological 
examination  samples  from  the  water-supply  of  a  city  may  be 
drawn  from  the  faucet,  but  the  water  should  first  be  allowed  to 
run  for  half  an  hour  or  longer.  From  other  sources  the  supply 
should  be  collected  in  sterilized  tubes  or  bottles,  taking  care  to 
avoid  contamination.  These  samples  should  be  examined  as 
promptly  as  possible,  for  the  water  bacteria  increase  rapidly 
in  number  after  the  samples  have  been  collected.  When  trans- 
portation to  some  distance  is  unavoidable  the  samples  should 
be  packed  in  ice,  but  even  this  precaution  does  not  preserve  the 
original  bacteriological  condition  of  the  water  at  the  time  of 
collection;  for  more  or  less  change  probably  takes  place  at  all 

1  U.  S.  Dep.  Agriculture,  Bu.  Plant  Ind.  Bulletin  64,  1904. 
8  Journ.  Inf.  Diseases,  Sup.  No.  2,  Feb.,  1906,  pp.  175-204. 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  183 

temperatures.  If  the  temperature  is  too  low,  and  the  water 
freezes,  more  or  less  of  the  bacteria  may  be  killed;  if,  on  the 
contrary,  the  temperature  is  not  low  enough  there  will  be  a 
multiplication  of  the  bacteria  in  transit.  Special  containers 
with  provision  for  packing  in  cracked  ice  should  be  employed 
for  shipment,  and  even  then  any  considerable  delay  should  be 
avoided. 

The  number  of  bacteria  may  be  determined  by  making  plates  of 
a  definite  quantity  of  the  water  with  gelatin  or  agar. 1  The  amount 
examined  ordinarily  is  i  c.c.  When  the  number  of  bacteria  is 
very  large,  a  smaller  quantity  must  be  taken,  and  it  may  be  neces- 
sary to  dilute  the  sample  ten  times  or  more  with  sterilized  water. 
The  amount  should  be  measured  with  a  sterilized,  graduated 
pipette.  The  water  is  mixed  with  melted  gelatin  or  agar  in  a 
tube  which  has  been  allowed  to  cool  after  melting.  After 
thorough  mixing,  remove  the  plug,  burn  the  edge  of  the  tube  in 
the  flame,  hold  in  a  nearly  horizontal  position  until  cool  and  pour 
into  a  sterilized  Petri  dish;  or  better,  measure  the  water  into 
the  Petri  dish,  and  pour  the  melted  medium  in,  and  mix.  The 
number  of  colonies  may  be  counted  on  the  third  or  fourth  day; 
the  later  the  better,  as  some  forms  develop  slowly  and  may  not 
present  visible  colonies  for  several  days;  but  the  plates  are  often 
spoiled  after  three  or  four  days  by  the  profuse  surface  growths  of 
certain  forms,  or  by  the  rapid  liquefaction  of  gelatin,  if  that  be 
used.  The  number  of  colonies  that  develop  is  supposed  to  repre- 
sent the  number  of  individual  bacteria  contained  in  the  quantity 
measured.  That  will  probably  not  always  be  the  case,  however, 
as  colonies  may  develop  from  a  clump  of  bacteria  which  have 
not  been  separated  from  one  another  by  the  mixing  process. 
Abbott  has  shown  that  the  number  of  colonies  is  usually  larger 
on  gelatin  plates  than  upon  agar  plates,  and  at  the  room  tempera- 
ture than  in  the  incubator.  This  observation  illustrates  the  fact 

1  For  standard  methods  of  water  analysis  see  the  report  of  a  Committee  of  the 
American  Public  Health  Association,  Journ.  Inf.  Diseases,  Supplement  No.  i,  May, 
1905;  also  Report  of  Committee,  American  Public  Health  Association,  New  York, 
1912. 


1 84 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


that  there  are  doubtless  many  kinds  of  bacteria  that  do  not  find 
favorable  conditions  for  development  on  ordinary  culture- 
media.  The  reaction  of  the  medium  has  an  important  influence 
upon  the 'development  of  these  water  bacteria  in  plate  cultures. 


FIG.  83. — Jeffer's  plate  (Bausch  and  Lomb}.     For  counting  colonies  of  bacteria  on 
circular  plates.    The  area  of  each  division  is  one  square  centimeter. 

When  the  number  of  colonies  is  small,  there  is  no  difficulty 
in  counting  them  as  they  appear  in  the  ordinary  Petri  dish. 
When  the  number  is  large,  some  kind  of  mechanical  device  may 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  185 

be  used  to  assist  counting.  The  Wolffhiigel  plate  is  a  large 
square  of  glass  resting  in  a  wooden  frame  painted  black.  The 
glass  plate  is  ruled  in  squares.  It  was  designed  particularly 
with  reference  to  the  form  of  plate-cultures  first  made  by  Koch. 
The  Petri  dish,  however,  may  be  placed  upon  the  glass  plate  and 


FIG.  84. — Surface  divided  in  square  centimeters  for  counting  colonies. 

the  cross  lines  be  used  to  assist  in  counting.  Lafar,  Pakes  and 
Jeffer  recommend  a  surface  painted  black,  ruled  with  white  lines 
which  represent  the  radii  of  a  circle,  which  may  be  still  further 
subdivided  by  other  lines.  Many  find  counting  easier  when  a 
black  surface  divided  into  squares  is  employed.  An  ordinary 


1 86  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

card  with  a  smooth  black  surface  divided  into  squares  by  white 
lines  may  be  placed  under  a  Petri  dish  and  will  be  found  to  serve 
very  well.  For  the  mere  examination  of  the  colonies  no  better 
surface  can  be  devised  than  the  ferrotype  plate  used  by  pho- 
tographers. The  examination  of  the  colonies  will  be  easier  if  a 
small  hand-lens  be  used.  Care  must  be  taken  not  to  mistake 
air-bubbles  or  particles  of  dirt  for  colonies  of  bacteria. 

In  any  case,  if  possible,  all  the  colonies  in  the  plate  should  be 
counted.  But  if  this  is  not  possible,  the  number  contained 
within  several  squares  may  be  counted  and  the  average  taken; 
knowing  the  size  of  the  squares  and  the  area  of  the  plate,  the 
number  contained  in  the  whole  plate  may  be  calculated. 

The  plating  may  be  done  by  rolling  the  medium  after  the 
manner  of  Esmarch.  When  the  number  of  colonies  is  not  large 
this  may  serve  very  well.  Counting  may  be  assisted  by  drawing 
lines  with  ink  on  the  outer  surface  of  the  test-tube.  It  is  obvious 
that  the  character  of  the  bacteria  is  of  prime  importance;  that 
pathogenic  organisms  may  occasionally  be  present,  even  when 
the  number  of  bacteria  to  the  cubic  centimeter  is  small.  But 
knowing  the  number  usually  found  in  a  good  water-supply,  any 
sudden  variation  above  that  number  is  to  be  looked  upon  with 
suspicion,  as  indicating  a  possible  contamination. 

The  bacteriological  examination  should  always  be  accom- 
panied by  a  chemical  examination,  and  by  an  inspection  of  the 
surroundings.  A  large  number  of  bacteria  is  to  be  expected 
when  the  water  has  been  subjected  to  unusual  agitation  from 
winds  or  currents  which  stir  up  the  bacteria  from  the  bottom. 

The  Detection  of  Intestinal  Bacteria. — Bacillus  coli  is  the 
organism  ordinarily  sought  as  a  proof  of  pollution  of  a  water- 
supply.  Various  quantities  of  the  water,  o.oi  c.c.,  o.i  c.c.  and 
i  c.c.  may  be  inoculated  into  three  series  of  fermentation  tubes 
containing  glucose  broth  or  lactose  bile.  These  are  incubated 
at  39°  C.  Plates  are  made  from  the  tubes  in  which  gas  is  pro- 
duced and  pure  cultures  obtained  from  the  colonies  for  further 
study  and  identification.  The  number  of  tubes  in  which  gas  is 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  187 

produced  is  regarded  as  presumptive  evidence  of  the  approxi- 
mate number  of  organisms  of  the  B.  coll  type  in  the  respective 
volume  of  water. 

The  recognition  of  pathogenic  bacteria,  such  as  the  germs  of 
typhoid  fever  and  Asiatic  cholera,  in  water  supplies  has  been 
accomplished  very  infrequently.  Search  for  the  cholera  germ 
is  best  undertaken  by  adding  a  large  volume,  i  to  10  liters,  of  the 
suspected  water  to  one-tenth  its  volume  of  a  sterile  solution  con- 
taining 10  per  cent  of  peptone  and  5  per  cent  of  sodium  chloride. 
After  incubation  for  twelve  to  twenty-four  hours  at  37°  C.,  trans- 
fers are  made  from  the  surface  of  this  culture  to  tubes  or  flasks 
containing  Dunham's  solution  (peptone  i  per  cent,  salt  0.5  per 
cent).  At  the  same  time  gelatin  plates  are  inoculated  from 
this  surface  material.  The  cholera  organism,  if  present,  tends 
to  outgrow  all  other  bacteria  in  the  surface  film  of  such  cultures, 
and  after  one  or  two  transfers  in  series  it  will  so  predominate 
that  it  may  be  recognized  by  specific  agglutination  with  a  cholera- 
immune  serum  in  high  dilution  (i-iooo).  The  appearance  of 
the  colonies  on  the  gelatin  plates  is  valuable  as  confirmatory 
evidence,  and  from  them  perfectly  pure  cultures  may  be  obtained 
for  further  study.  The  search  for  the  typhoid  bacillus  in  water 
is  usually  rather  hopeless.  It  has  occasionally  been  detected 
by  plating  the  water  on  special  media  such  as  litmus  lactose 
agar  or  gelatin,  or  by  culture  in  broth  at  39°  C.  and  inoculation 
of  guinea-pigs  or  white  rats  with  the  culture,  and  subsequent 
plating  of  the  heart's  blood  from  the  dead  animal.  Supposed 
typhoid  bacilli  isolated  in  this  way  must  satisfy  the  biochemical 
tests  for  B.  typhosus  and  furthermore  must  show  specific  agglu- 
tination with  high  dilutions  (i -100)  of  typhoid-immune  serum. 

If  it  is  not  already  apparent  from  what  has  been  said,  it  must 
be  here  emphasized  that  the  difficulty  of  detecting  the  presence 
of  pathogenic  bacteria  in  water  is  very  great,  and  the  length  of 
time  necessarily  consumed  in  making  the  tests  greatly  lessens 
the  value  of  the  results  when  obtained.  Added  to  this  is  the 
further  limitation  of  the  value,  that  a  negative  result,  i.e.,  where 


1 88  GENERAL  BIOLOGY    OF   MICRO-ORGANISMS 

no  pathogenic  bacteria  are  found,  cannot  be  taken  as  proof  that 
the  water-supply  under  examination  may  not  be  contaminated 
at  times.  Flugge1  has  shown  that  the  chemical  examination 
of  itself  also  permits  of  no  definite  conclusion  as  to  the  potability 
of  water.  It  would  seem  that  those  best  suited  by  training  and 
experience  and  who  are  capable  of  forming  disinterested  opinion 
attach  but  limited  importance  to  the  result  of  laboratory  exami- 
nations of  water  unaccompanied  by  a  sanitary  inspection.  In 
fact,  many  of  those  who  have  made  disinterested  study  of  the 
subject  are  inclined  to  question  the  value  of  the  ordinary  chemical 
and  bacteriological  water  analysis  in  toto,  and  in  view  of  the 
arbitrary  and  mechanical  manner  in  which  the  results  of  these 
analyses  are  sometimes  interpreted,  this  attitude  is  justified. 
It  would  seem,  however,  that  after  the  establishment  of  normal 
standards  for  a  given  locality,  such  analyses  are  useful  if  they 
are  checked  by  intelligent  consideration  of  all  the  conditions 
entering  into  the  case,  but  no  hard  and  fast  rules  can  be 
applied. 

Ice. — The  bacteriological  examination  of  ice  differs  in  no 
respect  from  that  of  water.  Although  development  may  be 
arrested,  the  vitality  of  bacteria  is  not  necessarily  impaired  by 
freezing.  Prudden  found  the  bacillus  of  typhoid  fever  alive 
in  ice  after  more  than  one  hundred  days.  However,  Sedgwick 
and  Winslow  have  stated  that  when  typhoid  bacilli  are  frozen 
in  water  the  majority  of  them  are  destroyed.3  Nevertheless, 
it  is  as  necessary  to  have  the  source  from  which  ice  is  taken  as 
carefully  scrutinized  as  that  of  the  water-supply,  especially  in 
view  of  the  universal  habit  of  cooling  water  in  the  summer  time 
by  adding  ice  directly  to  the  water.  It  is  better  to  cool  water 
and  articles  of  food  by  surrounding  with  ice  the  vessels  containing 
them. 

1  Flugge:  Zeitschrift  fur  Hygiene,  Bd.  XXII,  1896,  pp.  445  et  seq: 

2  Bolton:  Sanitary    Water    Supplies    for    Dairy    Farms.     Public    Health    and 
Marine  Hospital  Service,  Bulletin  41,  February,  1908,  p.  534. 

3  Clark.     Bacterial  Purification   of   matter   by  Freezing.      Reports   American 
Public  Health  Association,  Vol.  XXVII.     See  also  Hutchings  and  Wheeler:  Ameri- 
can Journal  Medical  Sciences,  Vol.  CXXVI,  p.  680. 


THE  DISTRIBUTION  OF  MICRO-ORGANISMS  189 

MICRO-ORGANISMS  OF  FOOD. 
\ 

Milk. — Milk  is  the  natural  food  of  young  mammals,  and 
naturally  it  is  taken  directly  from  the  mammary  gland  into 
the  digestive  tract  of  the  young  mammal.  For  many  centuries, 
however,  the  milk  of  certain  animals  has  been  extensively  used 
as  a  commercial  food  for  man.  The  principal  animals  furnishing 
commercial  milk  are  the  cow,  goat  and  mare.  The  chemical 
composition  of  milk  is  different  in  different  animals,  in  the  same 
animal  at  different  periods  of  lactation,  and  even  that  obtained 
at  different  stages  of  a  single  milking  shows  considerable  varia- 
tion. In  general  cow's  milk  has  the  following  composition. 

Variation.     Average. 

Fat 3-6  4  per  cent. 

Lactose 1-3  2  per  cent. 

Protein 5-8  7  per  cent. 

Water 84-88  87  per  cent. 

It  is  an  excellent  medium  for  the  growth  of  most  bacteria  and  is 
commonly  used  in  the  laboratory  for  this  purpose. 

There  are  about  200  species  and  varieties  of  bacteria  which 
commonly  occur  in  milk.  They  are  derived  in  part  from  the 
udder  itself.  Bacteria  are  always  present  in  the  milk  ducts  of 
the  udder  and  are  fairly  abundant  in  the  first  portions  of  milk 
drawn,  so  that  milk  very  carefully  drawn  from  healthy  animals 
may  contain  200  to  400  bacteria  per  cubic  centimeter.  Milk 
from  diseased  udders  may  be  very  rich  in  pathogenic  micro- 
organisms. As  the  milk  is  drawn,  many  micro-organisms  usually 
gain  entrance  to  it  from  the  atmosphere,  the  hands  of  the  milker 
and  the  utensils  with  which  it  comes  in  contact.  From  the 
body  of  the  cow,  particles  of  dust  and  hairs  drop  into  the  milk, 
carrying  with  them  the  flora  of  the  intestine  and  of  the  skin  of 
the  cow.  From  the  milker,  the  material  on  the  hands  and  possibly 
also  from  the  nose  and  mouth  may  reach  the  milk.  The  utensils, 
unless  sterilized  before  use,  contribute  the  microbic  flora  of  the 
previous  milkings,  of  the  water  used  for  cleansing  and  from  the 


1 90  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

person  who  handles  them.  From  the  air,  the  milk  may  receive 
further  contamination  (i)  from  flies  coming  to  drink  or  perhaps 
to  drown  without  a  clean  bill  of  health  from  their  port  of  last 
departure,  (2)  from  particles  suspended  as  dust  and  containing 
micro-organisms  derived  from  manure,  from  hay  and  straw, 
and  from  soil,  and  (3)  moist  droplets  expelled  from  the  mouth 
and  nose  of  the  milkers  and  of  the  cattle.  The  subsequent 
handling  of  the  milk  may  add  further  kinds  of  bacteria  from 
human  sources.  Modern  dairy  practice  in  vogue  in  the  produc- 
tion of  the  higher  grades  of  milk  eliminates  some  of  these  sources 
of  contamination  and  minimizes  the  importance  of  the  rest,  but 
nevertheless  fresh  milk  of  even  the  better  grades  contains  a 
great  variety  of  micro-organisms,  and  often  as  many  as  10,000 
to  100,000  per  cubic  centimeter  when  it  leaves  the  producer's 
dairy. 

The  usual  milk  flora  derived  from  these  various  sources  may 
be  classed  under  the  following  heads: 

A.  Lactic  acid  bacteria. 

1.  Bacterium  (streptococcus?)  acidi  lactici 

2.  Bacillus  coli  and  B.  lactis  aerogenes. 

3.  Long  rods  of  B.  bulgaricus  type. 

4.  Streptococcus  pyo genes. 

5.  Micrococcus  acidi  lactici. 

6.  Acid  formers  which  liquefy  gelatin. 

B.  Gelatin-liquefying  bacilli. 

7.  Rapidly  liquefying  types — B.  subtilis. 

8.  Slowly  liquefying  types. 

C.  Pigment-forming  bacteria. 

D.  Anaerobic  bacteria — B.  welchii,  putrefactive  anaerobes. 

E.  Special    types    causing   peculiar    fermentations,    such    as 
slimy  consistency,  bitter  taste  and  peculiar  odors. 

F.  Pathogenic  organisms — typhoid,    tuberculosis,    scarlatina, 
diphtheria,  diarrhea,  septic  sore  throat,  foot-and-mouth  disease, 
dysentery. 

G.  Other  fungi — Molds,  Oidia,  Yeasts,  Actinomyces. 


THE   DISTRIBUTION   OF   MICRO-ORGANISMS  IQI 

The  development  of  these  various  microbes  in  the  milk 
depends  very  much  upon  the  temperature  at  which  it  is  kept. 
At  o°  to  10°  C.  the  acid-forming  bacteria  grow  very  slowly  or 
not  at  all,  and  the  milk  may  remain  practically  unchanged  for 
many  days  or  even  weeks.  Eventually  some  of-  the  liquefying 
bacilli  or  the  slime-producing  types  may  gain  the  upper  hand 
and  change  the  consistency  and  flavor.  Between  10°  and  21° 
the  Bad.  acidi  lactici  is  almost  certain  to  gain  the  dominance 
and  rapidly  to  suppress  the  other  types,  and  it  produces  the 
normal  souring  of  milk.  Between  21°  and  35°  C.  the  organisms 
of  the  B.  coli  and  B.  lactis  aero  genes  groups  are  likely  to  pre- 
dominate and  at  temperatures  from  37°  C.  to  40°  C.  the  B.  bul- 
garicus  is  likely  to  gain  the  ascendency,  after  a  few  days  at 
any  rate.  These  may  be  regarded  as  the  normal  fermentations 
of  unheated  milk  of  very  good  quality.  The  other  microbes  in 
the  milk  are  not  destroyed  by  these  fermentations  but  their 
development  is  usually  held  in  check  somewhat. 

Shortly  after  the  coagulation  of  the  milk,  which  occurs  when 
the  lactic  acid  reaches  a  concentration  of  about  0.45  per  cent, 
the  living  bacteria  begin  to  diminish  in  number,  and  gradually 
Oidium  lactis  and  other  molds  become  prominent,  although  acid- 
resisting  forms  such  as  B.  bulgaricus  still  continue  to  grow. 
Organisms  of  these  kinds  seem  to  be  specially  concerned  in 
the  ripening  of  acid  curd  in  cheese  making.  Finally  the  acidity 
may  disappear  as  a  result  of  the  activity  of  molds,  and  putre- 
factive bacteria  find  the  opportunity  to  develop. 

If  the  milk  be  pasteurized,  the  bacteria  which  form  lactic 
acid  are  killed,  and  when  fermentation  occurs  it  is  likely  to  be 
different  from  the  normal  souring.  At  a  high  temperature, 
the  stormy  butyric-acid  fermentation  due  to  B.  welchii  may  be 
observed.  At  a  lower  temperature,  a  slow  putrefaction  due  to 
spore-forming  putrefactive  anaerobes  in  conjunction  with  other 
bacteria  may  occur.  These  fermentations  are  ordinarily  inhib- 
ited by  the  lactic  acid  produced  in  the  normal  souring  of  milk. 

Alcoholic  fermentation  of  milk  occurs  as  a  rule  only  when 


IQ2  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

special  ferments  are  purposely  added  to  produce  this  result. 
Kumyss  and  Kefir  are  fermented  milks  produced  in  this  way. 
The  starter  or  ferment  contains  yeasts  as  well  as  bacteria. 

The  pathogenic  micro-organisms  in  milk  are  derived  in  part 
from  unhealthy  cows — tuberculosis,  foot-and-mouth  disease, 
septic  sore  throat  (?) — but  in  a  larger  measure  from  the  people 
who  handle  the  milk  or  from  utensils — tuberculosis,  typhoid 
fever,  scarlatina,  diphtheria,  diarrheas,  dysentery,  septic  sore 
throat  (?).  The  bacteria  of  typhoid  fever,  diphtheria  and  dysen- 
tery are  known  to  multiply  in  milk.  The  microbes  of  tuberculo- 
sis and  foot-and-mouth  disease  may  persist  in  butter  and  cheese 
for  several  weeks  at  least. 

Leaving  out  of  consideration  the  question  of  specific  patho- 
genic micro-organisms,  the  presence  of  more  than  500,000  bac- 
teria per  cubic  centimeter  in  the  milk  regularly  fed  to  infants 
and  young  children  is  undoubtedly  harmful,  and  especially  so 
in  warm  weather.  Doubtless  many  factors  contribute  to  the 
causation  of  the  summer  diarrheas  and  the  summer  mortality 
of  children,  but  there  can  no  longer  be  any  question  that  a  milk 
rich  in  living  bacteria  as  food  for  these  children  is  one  of  the  very 
important  causes  of  their  illness  and  death. 

Milk  for  infant  feeding  should  come  from  clean,  healthy 
(tuberculin-tested)  cows,  should  be  handled  by  clean  healthy 
workmen,  in  clean  stables  and  rooms  and  with  clean,  sterilized 
utensils.  It  should  be  bottled  at  the  producing  dairy,  promptly 
chilled  to  10°  C.  or  below,  and  maintained  at  this  temperature 
until  delivered  at  the  home.  At  this  time  the  living  bacteria 
should  not  exceed  30,000  per  cubic  centimeter.  In  the  home, 
the  milk  should  be  kept  cold.  It  must  be  handled  only  with 
utensils  sterilized  by  boiling  in  water.  Boiled  water  is  employed 
in  making  the  necessary  dilutions.  If  the  milk  supply  is  not 
above  suspicion  the  milk  should  be  pasteurized  by  heating  to 
60°  C.  for  20  minutes.  The  dilution  is  prepared  and  filled  into 
separate  bottles  sufficient  in  number  so  that  one  may  be  used 
for  each  feeding  during  the  succeeding  24  hours.  Each  bottle 


THE   DISTRIBUTION    OF   MICRO-ORGANISMS  193 

is  chilled  in  cool  water,  then  ice  water,  and  finally  stored  in  the 
refrigerator.  Immediately  before  feeding  it  is  warmed  by  partial 
immersion  in  warm  water. 

Other  Foods. — Other  foods,  meats,  fish,  eggs,  vegetables 
and  fruits,  undergo  decompositions  due  to  more  or  less  definite 
types  of  micro-organisms,  and  the  activities  of  these  are  delayed 
or  prevented  by  modern  methods  of  preserving  foods,  in  some 
instances  very  successfully,  and  in  other  cases  with  limited  success.1 
Any  food,  and  especially  that  eaten  without  cooking,  may  serve 
as  a  passive  carrier  of  pathogenic  micro-organisms.  Salads, 
green  vegetables  and  fresh  fruits  may  undoubtedly  act  in  this 
way  during  epidemics.  Oysters  taken  from  sewage-polluted 
beds  have  been  found  to  convey  typhoid  fever.  Meats  derived 
from  mammals  may  contain  specific  germs  causing  disease  in 
both  animals  and  man,  such  as  tuberculosis,  anthrax  and  foot-and- 
mouth  disease.  The  flesh  of  bovine  animals  suffering  with 
enteritis  at  the  time  of  slaughter  seems  to  be  particularly  liable 
to  develop  poisonous  properties,  and  the  ill  effects  observed  in 
these  instances  may  have  been  due  to  a  specific  infection.  Para- 
typhoid fever  is  sometimes  traced  to  such  meat  as  a  cause. 

Meats  and  fish  are  rich  in  protein  and  their  decomposition 
by  saprophytic  bacteria  may  give  rise  to  various  poisonous  sub- 
stances, as  has  been  mentioned  on  page  170.  The  usual  course 
of  putrefaction,  however,  goes  on  without  very  strong  poisons 
being  produced,  as  we  may  judge  from  the  habitual  use  of  partly 
decomposed  foods  of  this  sort.  Virulent  poisons  are  occasion- 
ally encountered  and  some  of  these  are  due  to  the  presence  of 
specific  microbes,  B.  botulinus  of  Van  Ermengen,  B.  enteritidis  of 
Gaertner  and  the  paratyphoid  and  paracolon  bacilli.2 

1For  a  discussion  of  the  microbiology  of  foods  and  of  food  preservation  see 
MarchalFs  Microbiology  for  agricultural  and  domestic  science  students,  1911. 

2  Consult  Bolduan,  C.  F.:  Bacterial  Food  Poisoning,  N.  Y.  1909.  Also  Novy, 
F.  G.:  Food  Poisons,  Osier's  Modern  Medicine,  Vol.  I,  Phila.,  1907. 


CHAPTER  X. 
PARASITISM  AND  PATHOGENESIS. 

The  Parasitic  Relation. — The  presence  in  a  living  organism 
of  one  or  several  organisms  of  another  species,  which  live  as  para- 
sites upon  the  first,  is  a  phenomenon  of  common  occurrence  in  na- 
ture. Those  organisms  such  as  the  bacteria,  which  are  too  small 
to  harbor  visible  internal  parasites,  are  subject  to  the  parasitic 
ravages  of  larger  beings  such  as  amebae  and  other  protozoa, 
which  engulf  them  bodily  and  digest  them.  Man,  who  is  wont 
to  complain  of  his  parasitic  ailments,  takes  all  his  protein,  fat 
and  carbohydrate  from  the  bodies  of  plants  and  other  animals. 
Parasitism  in  the  larger  sense  is  a  well-nigh  universal  character- 
istic of  living  beings.  Parasitism  in  a  narrower  sense  usually 
applies  to  the  existence  of  a  smaller  organism,  the  parasite,  in 
or  on  the  body  of  a  larger,  the  host,  a  relation  in  which  the  host 
furnishes  the  parasite  its  necessary  food.  In  many  instances  the 
advantages  of  the  relation  are  wholly  one-sided,  but  in  others 
the  two  organisms  seem  to  be  of  mutual  benefit.  In  the  latter 
case,  the  condition  is  called  symbiosis.  The  infection  of  the 
roots  of  the  clover  with  Pseudomonas  radicicola,  which  promotes 
the  nitrogenous  nutrition  of  the  plant,  is  an  example  of  this  rela- 
tion. In  other  instances  the  two  organisms  living  in  close  associa- 
tion seem  neither  to  help  nor  injure  each  other.  They  are  then 
called  commensals  or  companions  at  the  same  table.  Internal 
parasites  occur  in  all  the  higher  animals  and  plants,  and  have 
been  found  even  in  the  bodies  of  protozoa.  Representatives  of 
all  the  great  classes  of  micro-organisms  are  found  among  the 
internal  parasites,  and  many  more  highly  organized  animals 
and  plants  also  lead  parasitic  lives.  Man,  alone,  is  subject  to 

194 


PARASITISM   AND   PATHOGENESIS  195 

infestation  with  parasitic  insects  and  numerous  worms,  in  addi- 
tion to  an  enormous  variety  of  microbes.  Whether  a  parasitk 
organism  is  to  be  regarded  as  a  symbiont,  a  commensal  or  a 
pathogenic  agent  depends  upon  the  effect  which  it  produces 
upon  its  host.  A  pathogenic  organism  is  one  whose  presence 
results  in  definite  injury  to  the  host. 

Pathogenesis. — In  human  pathology  the  phenomena  of  dis- 
ease have  for  centuries  been  the  object  of  careful  study  and 
speculation,  and  in  many  instances  the  phenomena  commonly 
associated  together  have  long  been  regarded  as  a  complex  result 
of  a  single  primary  cause,  and  the  condition  in  which  such  phe- 
nomena are  observed  has  been  regarded  as  a  single  morbid  en- 
tity or  a  definite  disease.  Even  the  most  ancient  records  indicate 
that  such  recognition  had  long  been  common  knowledge.  A 
beginner  in  parasitology  or  pathology  may  be  inclined  to  ascribe 
a  causal  relation  to  a  parasite  which  he  observes  in  the  body  of  a 
sick  individual;  in  fact  this  has  been  done  repeatedly.  The  log- 
ical requirements  for  the  proof  of  such  a  relationship  were  first 
formulated  by  Henle,  as  has  been  mentioned  in  the  historical 
sketch  in  the  introductory  chapter.  They  were  reformulated 
by  Koch,  who,  for  the  first  time,  was  able  to  comply  with  them 
in  respect  to  a  bacterial  disease.  They  may  be  stated  as  follows: 

1.  The  organisms  must  be  present  in  all  cases  of  the  particular 
disease. 

2.  The  organism  must  be  isolated  from  the  diseased  body 
and  propagated  in  pure  culture. 

3.  The  pure  culture  of  the  organism  when  introduced  into 
susceptible  animals  must  produce  the  disease. 

4.  In  the  disease  thus  produced,  the  organism  must  be  found 
distributed  as  in  the  natural  disease. 

Although  we  may  very  properly  consider  a  micro-organism  as 
the  probable  cause  of  a  disease  with  which  it  is  associated,  with- 
out satisfying  all  of  the  above  requirements,  the  experience  of 
the  last  three  decades  has  served  to  emphasize  more  and  more 


196  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

the  wisdom  of  reserving  final  judgment  wherever  these  rules  or 
similar  stern  logical  requirements  have  not  been  satisfied. 

Infectious  Disease. — An  infectious  disease  is  a  disease  due 
to  the  entrance  of  a  living  micro-organism  and  its  growth  in  the 
body.  Although  conservative  bacteriologists  are  sometimes 
loth  to  accept  a  disease  as  infectious  until  Koch's  rules  have 
been  satisfied,  most  are  agreed  that  a  disease,  which  can  be 
reproduced  indefinitely  by  the  inoculation  of  healthy  individuals 
in  series  with  material  taken  from  a  preceding  case,  is  due  to  a 
living  cause.  The  proof  that  a  disease  is  due  to  a  living  cause 
may  therefore  precede  the  identification  of  the  causal  organism, 
often  by  many  years. 

Possibility  of  Infection. — Whether  a  parasitic  organism  will 
be  able  to  enter  and  multiply  in  a  new  host  and  cause  disease 
depends  upon  a  number  of  circumstances,  the  most  important 
of  which  may  be  considered  under  four  heads,  namely,  the 
quality  of  the  microbe,  the  resistance  of  the  host,  the  quantity 
of  invading  parasites,  and  the  path  of  entrance.  The  course 
and  ultimate  result  of  an  infection  depend  also  to  a  marked 
degree  upon  these  same  factors.  In  general  the  qualifications 
of  the  micro-organism  depend  first  upon  the  experience  of  its 
ancestry  under  the  same  or  similar  enviromental  conditions, 
factors  inherent  in  its  species,  and  second,  upon  its  very  recent 
history,  factors  affecting  the  virulence  and  general  vigor  of  the 
individual  microbe.  Thus  the  tubercle  bacillus  is  qualified  by 
inheritance  for  a  parasitic  existence,  while  the  common  yeast  cell 
is  not.  Yet,  the  tubercle  bacillus,  when  cultivated  for  a  long  time 
on  artificial  media  may  lose  its  former  ability  to  grow  in  the 
animal  body.  The  factors  affecting  the  pathogenic  properties  of 
a  microbe  will  be  considered  in  the  succeeding  chapter. 

Susceptibility  and  Resistance. — Among  the  important  things 
in  the  nature  and  condition  of  the  host,  we  need  also  to  consider 
both  racial  and  individual  characters.  Certain  species  of  animals 
have  harbored  certain  parasites  for  so  long  that  the  latter  have 
become  adapted  to  growth  in  the  particular  species  of  host.  In 


PARASITISM   AND    PATHOGENESIS  1 97 

some  instances  the  adaptation  is  very  narrow  and  the  parasite 
may  be  able  to  exist  naturally  only  in  the  one  host  species,  as  for 
example  Spirochaeta  pallida.  Individual  resistance  of  different 
hosts  of  the  same  species  is  variable.  Age  is  one  important 
factor:  there  are  the  children's  diseases,  measles,  chicken- 
pox;  the  disease  of  active  adult  life,  pulmonary  tuberculosis, 
typhoid  fever;  and  the  diseases  of  the  aged,  pneumonia,  carci- 
noma. Hunger  and  thirst  have  been  shown  experimentally  to 
reduce  the  resistance  to  infection:  pigeons,  which  are  normally 
immune  to  anthrax  become  susceptible  when  starved.  The 
effect  of  fatigue  is  well  known:  a  white  rat,  normally  immune 
to  anthrax,  succumbs  to  it  after  prolonged  work  in  the  treadmill. 
Abnormal  chilling  of  hens  removes  their  immunity  to  anthrax 
and  abnormal  heating  of  frogs  affects  them  in  a  similar  way. 
Chemical  poisoning  also  increases  susceptibility  to  infection,  and 
cachexia  and  malnutrition  are  well-known  predisposing  factors 
to  such  infections  as  tuberculosis.  Traumatism  is  very  impor- 
tant, not  only  for  its  general  effect  upon  the  resistance  of  the  host, 
but  especially  in  the  reduction  of  local  resistance  by  destruction 
or  injury  of  tissue  (wounds).  There  are  certain  locations  where 
resistance  to  infection  is  naturally  lower,  such  as  the  ends  of 
growing  bones  and  the  interior  of  the  parturient  uterus. 

Number  of  Invaders. — The  quantity  of  infectious  material 
introduced  is  of  importance  in  determining  whether  infection 
will  or  will  not  occur.  Very  few  species  of  microbes  are  capable 
of  causing  disease  when  only  a  single  individual  organism  is  in- 
troduced into  the  body.  A  large  number  of  microbes  entering 
at  the  same  time  seems  to  overburden  the  defensive  powers  of 
the  body  so  that  some  of  the  parasites  succeed  in  establishing 
themselves  and  multiplying. 

Modes  of  Introduction. — There  are  various  avenues  by  which 
micro-organisms  may  enter  the  body  to  produce  disease.  In- 
fection of  the  ovum  in  the  ovary  with  spirochetes  and  protozoa 
is  known  to  occur  in  some  insects,  and  Rettger  has  shown  that 
this  phenomenon  occurs  in  the  hen  infected  with  Bacterium  pul- 


198  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

lorum.  The  human  ovum  also  seem  occasionally  to  be  infected 
with  Spirochata  pallida  in  this  way.  It  may  also  become  in- 
fected with  the  same  organism  derived  from  the  seminal  fluid. 
The  developing  fetus  is  sometimes  invaded  by  pathogenic  micro- 
organisms introduced  through  the  placental  circulation.  The 
organisms  of  tuberculosis,  small-pox,  typhoid  fever  and  the 
pyogenic  cocci  are  known  to  be  transmitted,  somewhat  uncom- 
monly to  be  sure,  in  this  way.  As  a  rule  the  germ  must  be  circu- 
lating in  the  blood  of  the  mother  in  considerable  numbers,  or 
there  must  be  actual  infectious  lesions  of  the  placenta  before 
placental  transmission  occurs.  After  birth  non-pathogenic  mi- 
crobes gain  access  to  the  entire  surface  of  the  body  and  penetrate 
the  various  canals  opening  to  the  exterior  to  certain  normal 
limits.  Pathogenic  germs  may  be  introduced  with  the  food  and 
drink,  which  is  the  common  natural  mode  of  infection  with  cholera 
and  typhoid  fever  in  man  and  with  tuberculosis  in  hogs  and  cattle. 
The  barrier  presented  by  the  activity  of  the  gastric  juice  is  fre- 
quently passed  in  safety  by  the  ingested  microbes.  Inhalation 
is  probably  the  most  common  way  in  which  tuberculous  infection1 
reaches  the  lungs  in  man,  although  there  is  conclusive  evidence 
that  tuberculosis  in  this  location  may  be  derived  from  the  alimen- 
tary tract  through  the  blood  stream.  Experimentally,  guinea- 
pigs  are  much  more  susceptible  to  infection  with  tubercle  bacilli 
by  inhalation  than  by  ingestion.  Mere  application  of  the  in- 
fectious agents  to  the  epithelial  surface  of  the  skin  or  mucous 
membranes  results  in  infection  in  many  instances  and,  indeed, 
infection  by  ingestion  and  inhalation  may  be  regarded  as  examples 
of  this.  The  mucous  membranes  of  the  urethra  and  the  eye,  and 
also  of  the  rectum  in  young  children,  are  especially  susceptible  to 
infection  with  the  gonococcus.  The  unbroken  skin  may  be  infected 
with  staphylococci,  which  seem  to  penetrate  through  the  hair  fol- 
licles and  sebaceous  glands,  giving  rise  to  boils  and  carbuncles;  but 
to  most  microbes  the  uninjured  skin  presents  an  effective  barrier. 

1McFadyean,  Journal  Royal  Institute  of  Public  Health,  1910,  Vol.  XVIII,  pp. 
703-724. 


PARASITISM  AND   PATHOGENESIS  1 99 

The  question  whether  infectious  agents  may  penetrate  epithe- 
lium and  gain  the  lymph  or  blood-vessels  beyond  without  causing 
a  local  lesion,  has  received  considerable  attention  and  it  seems 
to  be  established  as  certainly  possible  in  the  intestine  during 
the  absorption  of  fat,  and  it  may  perhaps  occur  in  other  locations. 

Infection  through  wounds,  even  minute  breaks  in  the  epithe- 
lial covering,  is  very  common.  Such  wounds  made  by  insects 
are  the  common  portals  of  entry  for  the  germs  of  malaria,  plague, 
yellow  fever,  relapsing  fever  and  many  more  diseases.  Larger 
wounds  nearly  always  become  infected  with  pyogenic  cocci 
unless  they  are  properly  cared  for.  The  introduction  of  infectious 
material  into  the  subcutaneous  tissue  may  occur  accidentally  in 
deep  wounds  and  is  a  common  mode  of  inoculation  in  the  labora- 
tory. Infection  with  the  anaerobic  bacillus  of  tetanus  frequently 
occurs  in  this  type  of  wound. 

Infections  of  the  peritoneal  cavity,  pleural  cavities  and  cavi- 
ties of  the  joints  result  from  penetrating  wounds,  by  the  entrance 
of  bacteria  from  contiguous  tissues,  as  through  the  intestinal 
wall  into  the  peritoneal  cavity,  and  through  the  blood  and  lymph 
channels. 

Local  Susceptibility. — The  invading  parasite  is  favored  by 
conditions  of  local  susceptibility  such  as  tissue  destruction, 
presence  of  necrotic  tissue  and  foreign  bodies,  and  also  by  the 
presence  of  other  infectious  microbes.  Small-pox  and  staphylo- 
coccus,  tetanus  and  the  pus  cocci,  scarlet  fever  and  streptococcus, 
are  common  examples  of  such  mixed  infections.  In  some  in- 
stances one  infection  predisposes  to  another.  For  example, 
measles  is  likely  to  favor  the  development  of  tuberculosis;  the 
caseous  tubercle  is  likely  to  be  invaded  by  the  streptococcus. 
These  subsequent  invasions  are  spoken  of  as  secondary  infections. 

Local  and  General  Infections. — The  invading  microbes  may 
remain  localized  near  the  point  of  entrance,  as  for  example  in 
tetanus  and  diphtheria.  In  such  cases  the  general  effects  may  be 
due  to  disturbance  in  function  of  the  local  tissue,  such  as  laryngeal 
obstruction  in  diphtheria,  or  to  the  absorption  into  the  lymph 


200  GENERAL  BIOLOGY    OF   MICRO-ORGANISMS 

and  blood  of  poisons  produced  at  the  infected  site.  Such  ab- 
sorption results  in  toxemia  with  symptoms  due  to  poisoning  of 
distant  tissue  elements.  On  the  other  hand,  the  infectious 
agent  may  pass  quickly  to  the  blood  stream  without  appreciable 
local  reaction  and  multiply  there,  as  in  malaria,  trypanosomiasis 
and  streptococcus  bacteremia.  Again  there  may  first  develop 
an  intense  local  reaction,  with  subsequent  extension  to  the 
blood  stream  with  fatal  issue,  as  in  malignant  pustule  (anthrax). 
In  other  instances  repeated  temporary  invasions  of  the  blood 
occur,  with  numerous  localized  abscesses  in  various  parts  of 
the  body,  a  condition  to  which  the  name  pyemia  has  been  applied. 
Of  particular  interest  are  those  general  infections  of  the  blood 
stream,  which  finally  fade  away,  but  leave  behind  localized 
infections  in  the  joints,  on  the  heart  valves,  in  the  central  nervous 
system,  or  other  parts  of  the  body.  Sleeping  sickness,  syphilis, 
acute  articular  rheumatism  and  generalized  gonococcus  infection 
belong  in  this  category. 

Transmission  of  Infection. — The  manner  in  which  an  infectious 
agent  passes  from  its  host  to  a  new  victim  varies  considerably. 
In  general  it  may  be  said  to  occur  (i)  by  direct  contact  or  close 
association,  transmission  by  contagion,  (2)  through  the  agency  of 
intermediate  dead  objects  as  passive  carriers,  transmission  by 
fomites,  or  (3)  through  the  agency  of  a  living  or  dead  object  in 
which  the  parasite  undergoes  development  or  multiplication, 
transmission  by  miasm.  These  terms  have  been  employed  in 
the  past  to  designate  rather  hypothetical  objects  not  to  say 
abstract  ideas,  and  their  application  to  the  facts  learned  by 
modern  research  is,  perhaps,  not  desirable.  Nevertheless,  they 
may  be  made  to  fit  the  observed  phenomena  in  a  way.  Thus, 
syphilis  and  gonorrhea  are  transmitted  by  contagion;  diphtheria 
and  small-pox  by  contagion  and  by  fomites;  tetanus  and  anthrax 
by  fomites  and  perhaps  also  miasm;  plague  by  contagion,  fomites 
and  miasm  (through  the  rat  and  flea);  malaria,  trypanosomiasis 
and  yellow  fever  by  miasm.  All  of  these  are  doubtless  infectious 
diseases  but  some  of  them  are  not  naturally  spread  by  contact  at 


PARASITISM   AND   PATHOGENESIS  2OI 

all.  In  studying  each  disease  it  will  be  necessary  to  consider 
the  avenues  by  which  the  parasite  leaves  the  patient,  its  existence 
in  the  external  world  and  the  means  of  gaining  access  to  its  new 
victim. 

Healthy  Carriers  of  Infection. — A  person  or  animal  may 
harbor  virulent  infectious  agents  without  showing  symptoms  of 
disease,  and  may  serve  as  a  source  of  infection  to  others.  This 
was  clearly  recognized  in  the  sixteenth  century  by  Fracastorius 
as  a  factor  in  the  spread  of  syphilis.  Only  recently  has  its 
importance  in  other  diseases  been  emphasized. 


CHAPTER  XI. 
THE  PATHOGENIC  PROPERTY  OF  MICRO-ORGANISMS. 

Adaptation  to  Parasitism. — In  order  to  live  as  a  parasite,  an 
organism  must  be  adapted  to  grow  under  the  conditions  met 
with  in  the  body  of  the  host,  but  in  order  to  produce  disease  it 
must  also  injure  the  host.  The  most  perfect  adaptation  of 
parasitism  is  probably  exhibited  by  those  micro-organisms 
which  do  not  injure  the  host,  the  symbionts  and  commensals, 
as  it  is  obviously  to  the  interest  of  the  parasite  to  keep  its  host 
alive.  An  adaptation  of  this  kind  usually  requires  that  the 
microbe  shall  either  grow  very  slowly,  or  shall  be  so  situated 
that  the  excessive  numbers  resulting  from  its  multiplication 
may  readily  pass  out  of  the  host  or  be  disposed  of  in  someway; 
otherwise  the  host  would  be  physically  crowded  out.  This  sort 
of  adaptation  is  illustrated  by  the  normal  intestinal  bacteria. 
Parasites  which  invade  the  tissues  of  the  body  rarely  show  such 
adaptation.  It  is,  perhaps,  approached  to  some  extent  by 
the  slow-growing  bacilli  of  leprosy  and  tuberculosis.  In  most 
instances  of  parasitism,  however,  there  is  more  or  less  of  a  struggle 
between  the  invader  and  the  host  for  the  possession  of  the  field, 
and  the  phenomena  of  disease  are  incident  to  this  combat. 

Virulence. — The  ability  of  the  parasite  to  injure  its  host,  is 
designated  as  virulence.  Virulence  depends  in  part  upon  growth 
vigor,  but  also  upon  other  factors  largely  unknown.  A  great 
deal  is  known  about  specific  methods  of  changing  the  virulence 
of  micro-organisms,  and  various  procedures  are  commonly  em- 
ployed with  this  object  in  view.  A  diminution  in  virulence  is  called 
attenuation  and  an  increase  in  virulence,  exaltation.  Attenua- 
tion was  first  observed  by  Pasteur  in  a  culture  of  Bacterium 
amsepticum  (chicken  cholera)  grown  in  broth  in  the  presence  of 

202 


PATHOGENIC   PROPERTY   OF    MICRO-ORGANISMS  203 

air.  Pneumococci  and  streptococci  also  attenuate  rapidly  in 
artificial  culture.  Even  those  bacteria  which  retain  then- 
virulence  in  ordinary  cultures  become  attenuated  when  grown  at 
unusually  high  temperatures  (42°  C.)  or  in  the  presence  of 
antiseptics,  both  of  which  methods  have  been  employed  in 
attenuating  the  anthrax  bacillus.  Attenuation  also  results 
sometimes  from  parasitism  in  hosts  of  another  species.  Variola 
and  vaccinia  present  a  conspicuous  example  of  this.  Mere 
dessication  of  a  virus  seems  to  attenuate  it  in  some  instances 
(rabies)  but  this  is  somewhat  doubtful.  Many  pathogenic 
agents  become  somewhat  attenuated  upon  long  residence  in  the 
same  host  in  chronic  infections.  Exaltation  of  a  virus,  on  the 
other  hand,  is  accomplished  by  rapid  passage  through  susceptible 
animals  in  series.  When  the  organism  is  too  attenuated  to 
produce  an  infection  alone,  it  may  be  aided  by  the  admixture  of 
other  organisms  (mixed  infection)  or  by  the  presence  of  irritating 
foreign  bodies  (splinters,  stone  dust )  or  by  mechanical  protection 
in  collodion  capsules. 

Microbic  Poisons. — The  weapons  which  the  pathogenic 
agent  employs  to  injure  its  host  are  various.  The  physical 
mass  of  the  invaders  may  be  injurious,  more  particularly  by 
obstructing  blood-vessels,  as  in  estivo-autumnal  malaria  in  man 
and  anthrax  in  the  mouse.  Usually,  however,  the  offensive 
weapons  are  chiefly  chemical  poisons.  The  soluble  toxins,  or 
true  toxins  are  substances  of  unknown  chemical  composition 
produced  inside  bacterial  cells  and  passed  out  to  their  surround- 
ings. These  so-called  extracellular  toxins  include  the  most 
poisonous  substances  known.  Brieger  and  Cohn  obtained  a 
toxin,  still  impure,  from  tetanus  bacilli,  of  which  five  one  hundred 
million ths  of  a  grams  (.00000005  gram)  killed  a  mouse  weighing 
15  grams.  At  this  rate  .00023  of  a  gram  would  kill  a  man  weigh- 
ing 70  Kilos.1  The  soluble  toxins  elaborated  by  the  diphtheria 
and  tetanus  bacilli  have  been  studied  most,  and  many  of  our 
ideas  concerning  toxins  in  general  have  been  derived  from  these 

1  Vaughan  and  Novy,  Cellular  Toxins,  Phila.,  1902,  p.  62. 


2O4  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

studies.  These  poisons  are  rapidly  destroyed  by  heat,  resembling 
enzymes  in  this  respect.  They  differ  from  enzymes  in  that 
they  are  used  up  in  combining  with  tissue.  Thus  tetanus  toxin 
may  be  completely  neutralized  by  the  addition  of  brain  tissue, 
and  either  diphtheria  or  tetanus  antitoxin  may  be  quantitatively 
neutralized  by  its  specific  antitoxin.  Ehrlich  in  his  study  of  the 
reactions  of  diphtheria  toxin  showed  that  on  standing  it  loses 
much  of  its  poisonous  property  without  any  diminution  in  its 
ability  to  combine  with  diphtheria  antitoxin,  and  to  this  less 
poisonous  substance  he  gave  the  name  toxoid.  From  this  observa- 
tion he  concluded  that  the  toxin  molecule  contains  at  least  two 
very  definite  atomic  groups.  One  of  these  is  comparatively 
stable  and  serves  for  attachment  of  the  toxin  molecule  to  the 
cell  attacked  by  it,  and  is  called  the  haptophorous  group  or 
simply  haptophore.  The  other  recognizable  chemical  group 
disintegrates  more  readily  and  is  that  which  bears  the  poisonous 
property.  To  this  he  gave  the  name  of  toxophorous  group  or 
toxophore.  In  their  reactions  toxins  behave  in  part  like  feebly 
dissociated  chemical  compounds,  as  has  been  shown  by  Arrhenius 
and  Madsen,  but  the  reactions  by  which  they  combine  are  only 
slightly  or  not  at  all  reversible  and,  moreover,  take  place  in 
variable  proportions.  Bordet  very  aptly  compares  the  reactions 
of  toxin  to  the  union  of  a  dye  with  a  stainable  material.  Bacteria 
also  produce  poisons  which  are  part  of  their  own  body  substance, 
and  set  free  only  upon  their  death  and  disintegration.  These 
are  spoken  of  as  intracellular  toxins.  Injurious  substances  may 
also  be  produced  from  the  tissue  of  the  host  by  a  secondary  action 
outside  the  cell  of  the  parasite,  but  these  secondary  products 
play  a  very  minor  role. 

Defensive  Mechanisms. — The  defensive  armor  of  parasites 
seems  also  to  be  in  part  physical  and  in  part  chemical,  and  perhaps 
we  may  regard  the  physiological  adaptation  to  slow  growth  as  a 
defensive  mechanism  because  it  tends  to  avoid  exciting  the 
opposition  of  the  host.  The  physical  structure  seems  to  be 
protective  in  case  of  the  waxy  bacteria  (tubercle  and  leprosy) 


PATHOGENIC   PROPERTY    OF   MICRO-ORGANISMS  205 

and  the  capsules  of  other  bacteria  may  serve  a  similar  purpose 
(pneumococcus).  There  is  some  indication  that  micro-organisms 
may  produce  special  chemical  substances  to  neutralize  the 
agencies  which  the  host  employs  against  them.  These  defensive 
substances  have  been  designated  by  Bail  as  aggressins.  Ehrlich 
has  also  found  evidence  of  the  acquirement  of  immunity  to 
chemical  substances  by  certain  pathogenic  microbes,  especially 
trypanosomes  and  spirochetes,  and  he  ascribes  this  property  of 
the  parasites  to  an  alteration  of  their  cell-chemistry. 


CHAPTER  XII. 
REACTION  OF  THE  HOST  TO  INFECTION. 

Facts  and  Theories. — The  host  reacts  to  the  presence  of  a 
pathogenic  agent  by  a  number  of  alterations  in  its  physiological 
activities.  Some  of  these  alterations  are  gross  and  well  known 
as  the  clinical  manifestations  of  an  infectious  disease;  others 
require  special  search  for  their  detection;  while  some,  doubtless 
a  considerable  number,  still  pass  unobserved.  Many  of  these 
changes  are  susceptible  of  very  accurate  observation,  and  in 
most  instances  the  observed  facts  are  well  established.  A  clear 
understanding  of  the  relation  of  the  various  facts  to  each  other 
involves  some  imaginative  reasoning,  and  various  hypotheses 
have  been  advanced  to  explain  the  phenomena  observed,  and  to 
fill  in  the  gaps  in  our  knowledge.  The  student  may  need  to  be 
on  his  guard  not  to  confuse  facts  susceptible  of  observation  with 
hypothetical  deductions  based  upon  such  observations.  Both 
have  their  peculiar  value.  An  understanding  of  the  phenomena 
of  pathological  physiology  must  be  based  upon  correct  ideas  of 
normal  physiology  and  accurate  knowledge  has  not  fully  replaced 
hypothesis  in  this  latter  field. 

Physiological  Hyperplasia. — Under  normal  conditions  each 
cell  of  the  human  body  is  in  close  association  with  other  cells 
and  with  the  body  fluids,  and  is  subject  to  the  physical  and 
chemical  stimulation  of  cells  and  fluids.  One  of  the  effects  is 
apparently  to  restrain  the  prolif  erative  activity  of  the  cells.  When 
certain  of  these  cells  are  destroyed,  or  even  certain  parts  of  them, 
this  restraint  is  removed,  and  the  natural  tendency  to  prolifera- 
tion asserts  itself,  resulting  in  the  production  of  new  cells  or  of 
new  parts  to  replace  the  old,  and  usually  more  than  compensates 
for  the  loss.  This  somewhat  hypothetical  conception,  due  to 

206 


REACTION   OF   THE   HOST    TO   INFECTION  207 

Carl  Weigert,  serves  to  explain  tissue  hyperplasia  and  repair 
following  exercise  or  local  destruction  of  tissue.  Examples  of 
these  phemomena  will  occur  to  the  reader. 

Phagocytosis  and  Encapsulation. — The  mere  physical  mass  of 
a  parasite  within  the  tissue  acts  as  a  foreign  body  and  it  becomes 
surrounded  by  tissue  elements.  If  it  is  minute,  certain  cells  of 
the  body  (phagocytes)  flow  around  and  ingest  it,  as  was  first 
observed  by  Metchnikoff.  If  it  is  larger,  the  connective  tissue 
cells  proliferate  and  surround  it,  and  eventually  contract  into  a 
firm  capsule.  Further,  the  tissues  produce  enzymes  capable 
of  dissolving  many  foreign  substances  introduced  in  this  way 
(parenteral  digestion).  If  the  foreign  body  is  insoluble,  it  will- 
remain  encapsulated,  or,  if  sufficiently  minute,  it  may  be  trans- 
ported considerable  distances  inside  wandering  cells  and  eventu- 
ally be  deposited  in  a  lymph  gland.  The  wholly  passive  para- 
site or  the  dead  body  of  a  micro-organism  is  therefore  either 
digested  and  dissolved,  ingested  by  cells,  or  encapsulated  in 
fibrous  tissue.  Most  infectious  agents  are  not  passive  in  this 
way,  as  we  have  seen,  but  tend  actively  to  grow  and  multiply, 
to  absorb  and  utilize  food  material,  and,  most  important  of  all, 
to  produce  various  substances  which  stimulate  or  poison  the 
cells  of  the  host.  Against  these  the  physical  measures  of  inges- 
tion  (phagocytosis)  and  encapsulation  are  often  inadequate  de- 
fenses and  may  be  entirely  useless. 

Chemical  Constitution  of  the  Cell. — Ehrlich  has  compared 
the  living  body  cell  to  a  complex  chemical  molecule;  in  fact  it 
may  be  said  that  he  regards  the  living  cell  as  an  enormous  mole- 
cule, a  chemical  unit  of  great  complexity.  Certain  atom  groups 
within  this  molecule  are  pictured  as  relatively  very  stable  and 
they  constitue  the  chemical  nucleus  (not  to  be  confused  with 
the  anatomic  nucleus).  Grouped  about  this  chemically  stable 
center  are  very  many,  more  labile  atom  groups  which  readily 
enter  into  chemical  reaction  with  substances  in  the  surrounding 
medium.  The  conception  is  derived  directly  from  well-known 
facts  in  organic  chemistry.  For  example  when  benzoic  acid, 


2O8  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

CeHVCOOH,  reacts  with  other  chemicals  the  reaction  takes 
place  at  the  reactive  group,  or  side-chain,  rather  than  in  the 
nucleus.  The  graphic  formula  may  illustrate  this  point  better. 

H 

I 
H          C  O 

\    /\         II 
C         C— C— OH 

I 
H— C         C 

\/   \ 
C          H 

H 

The  six  carbon  atoms  in  the  ring  are  stable,  and  a  strong  chem- 
ical reagent,  such  as  phosphorus  pentachloride,  reacts  with  the 
side-chain  without  attacking  the  ring.  So  in  the  living  cell, 
Ehrich  assumes,  as  a  working  hypothesis,  the  existence  of  a 
wonderfully  complex  but  comparatively  stable  chemical  nucleus, 
with  abundant  and  various  more  reactive  side-chains.  These 
latter  serve  to  combine  with  food  materials  in  the  surrounding 
lymph,  and  these  are  then  utilized  in  the  cell  by  an  intramo- 
lecular rearrangement  of  atoms  which  is  always  in  progress.  Use- 
less atomic  groups  formed  in  the  metabolism  of  the  cell  are  de- 
tached and  passed  off  as  excretions.  These  reactions  of  intra- 
molecular rearrangement  and  molecular  disintegration  also 
find  their  analogues  in  carbocyclic  chemistry. 

Antitoxins. — Von  Behring  and  Kitasato  (1890-91)  showed 
that  animals  injected  with  small  non-fatal  doses  of  toxin  of  the 
tetanus  bacillus,  produce  as  a  result  of  this  treatment  a  some- 
thing which  circulates  in  solution  in  the  blood  plasma,  which 
is  capable  of  neutralizing  the  poisonous  properties  of  the  tetanus 
toxin.  Soon  afterward  von  Behring  obtained  analogous  results 
with  the  toxin  of  diphtheria.  The  protective  substances  in  the 
blood  were  called  antitoxins.  The  exact  chemical  composition 


REACTION   OF    THE   HOST   TO    INFECTION 


209 


of  these  substances  is  unknown.     They  accompany  the  pseudo- 

globulin  fraction  of   the  plasma  in  its  chemical  analysis,1  but 

the  union  here  is  probably  a  mere  physical  adsorption  or  very 

unstable  chemical  combination.     Ehrlich  explains  the  formation 

of  antitoxin  on  the  basis  of  his  side-chain  theory  as  follows. 

The  molecule  of  toxin  attacks 

the  body  cell  at   one   of   its 

side-chains  or  receptors  which 

is  best  adapted  to  this  reac- 

tion.    In  the  resulting  intra- 

molecular rearrangement  the 

toxin  reveals  itself  as  a  dis- 

turbing element,  causing  de- 

struction  of   that  portion  of 

the  cell  to  which  it  has  be- 

come attached.     In  recover- 

ing from  this  disturbance  the 

cell      overcompensates      by 

forming  an  excessive  number 

Of  the  particular  kind  of  side- 

r 

chain    destroyed,     and    some      can  Medical  Association,  1905,  p.  955.)     a, 

of  the  excess  side-chains  are 

detached,  and  circulate  in  the 

. 

blood,  ready  to  react  with 
toxin  entirely  apart  from  the  cell  which  has  produced  them. 
These  constitute  Ehrlich's  receptors  of  the  first  order  and  their 
sole  effect  upon  the  toxin  is  that  of  combining  with  it.  The  free 
receptors  circulating  in  the  blood  give  it  its  antitoxic  property. 

Precipitins.  —  Other  chemical  products  of  bacterial  growth 
are  attacked  and  rendered  insoluble  by  products  of  the  body 
cells.  Kraus2  (1897)  showed  that  animals  injected  with  cultures 
of  bacteria  produce  a  substance  or  substances,  which  circulates 
in  the  blood  and  is  capable  of  causing  a  precipitate  when  mixed 

1  Banzhaf,  Johns  Hopkins  Hospital  Bull.,  1911,  Vol.  XXII,  pp.  106-109. 

2  Wiener  klin.  Wochensckr.,  1897,  X,  p.  736. 

14 


.¥.IG-  85.-Receptor  of   the    first    order 
uniting  with  toxin.     (Journal  of  the  Amen- 


the  toxm  molecule;  e,  haptophore  of  the  cell 
receptor. 


210 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


with  the  clear  filtrate  of  the  cultures  of  the  same  bacteria.  The 
parenteral  introduction  of  any  foreign  protein  in  solution  stimu- 
lates' the  production  of  a  substance  which  will  precipitate  it.1 
These  substances,  which  are  called  precipitins,  resemble  enzymes 
in  many  respects.  Thus,  the  precipitin  produced  by  the  injec- 

tion of  a  milk,  causes  a 
change  in  the  milk  very 
similar  to  that  caused  by 
rennet.  Rennet,  however, 
coagulates  milk  from  vari- 
ous animals  while  the  milk 
precipitin  is  specific,  within 
certain  limits,  for  the  one 
kind  of  milk.  Precipita- 
tion results  only  when  the 
blood  serum  (precipitin)  is 
combined  with  the  proper 
amount  of  the  culture  fil- 
trate or  other  protein  *so- 

FIG.  86.—  Receptors  of  the  second  order  and  lution      (pretipitinogen)— 

some  substance  uniting  with  one  of  them.  (Jour-  wupn  tnn  lpro-p  an  PYPP^  of 

nal  of  the  American  Medical  Association,  1905,  p.  wnen  to°  large  an 

1113.)     c,  Cell  receptor  of  the  second  order;  d,  one  or  the  other  is  used  no 
toxophore  or  zymophore  group  of  the  receptor; 
e,  haptophore  of  the  receptor;  /,  food  substance 

or-r^duf  °/  Bacterial  disintegration  uniting 

the  haptophore  of  the  cell  receptor. 


-, 

wit 


.    . 
precipitate     occurs. 

H  h  explains  the  formation 
of  precipitins  on  the  basis 

of  his  side-chain  theory  in  the  same  way  as  the  production  of 
antitoxins  was  explained.  The  foreign  protein  stimulates  the 
body  cells  to  produce  specific  receptors  capable  of  combining 
with  it.  In  this  instance,  however,  the  receptor  not  only  com- 
bines with  the  foreign  material,  but  also  brings  about  a  definite 
change  in  it  which  is  evidenced  by  the  phenomenon  of  precipita- 
tion. The  side-chain  therefore  contains  at  least  two  distinct 
atomic  groups,  one  of  which  serves  to  combine  with  the  pre- 

1  Specific  precipitin  tests  have  been  employed  to  some  extent  in  determining 
the  source  of  blood  stains  and  of  meats.  See  Citron,  Immunity,  translated  by 
Garbat,  Phila.,  1912,  p.  112. 


REACTION   OF   THE   HOST   TO   INFECTION  211 

cipitinogen,  and  is  specific  in  nature,  and  another  which  brings 
about  the  change  evidenced  by  formation  of  the  precipitate^ 
The  former  of  these  chemical  groups  is  called  the  combining  or 
haptophorous  group  or  haptophore,  and  the  latter  is  called  the 
ferment-bearing  or  zymophorous  group  or  zymophore.  This 
type  of  side-chain  is  Ehrlich's  receptor  of  the  second  order.  It 
is  represented  in  the  figure  as  possessing  one  smooth  branch 
which  serves  for  simple  attachment,  the  haptophore,  and  one 
branch  equipped  with  saw-teeth  to  suggest  its  property  of  pro- 
ducing chemical  change,  the  zymophore.  The  precipitin  pres- 
ent in  the  blood  plasma  is  supposed  to  consist  of  such  receptors 
which  have  become  detached  from  the  cell  producing  them. 

Agglutinins. — Gruber  and  Durham  (1896)  found  that  the 
blood  of  animals  suffering  from  certain  infections  has  the  power 
of  causing  the  bacteria  involved  to  clump  together  and  lose  their 
motility  when  it  is  added  to  a  broth  culture  or  a  suspension  of  the 
bacteria  in  salt  solution.  The  phenomenon  has  been  observed 
in  connection  with  many  bacteria,  not  only  motile  but  also  non- 
motile  species,  but  the  most  important  examples  are  the  typhoid, 
paratyphoid,  cholera  and  dysentery  organisms.  In  typhoid  and 
paratyphoid  fever  the  agglutination  test  is  used  as  an  aid  in  diag- 
nosis of  the  disease  by  testing  patient's  serum  against  known 
cultures,  and  the  test  with  known  serum  is  important  in  the  iden- 
tification of  cultures  of  any  of  these  bacteria.  Agglutinins  are 
comparatively  stable  substances  although  they  decompose 
rapidly  at  70°  to  75°  C.  When  dried  they  keep  for  a  long  time. 
In  Ehrlich's  theory,  the  agglutinins  are  classed  as  receptors  of 
the  second  order,  along  with  the  precipitins. 

The  Phenomenon  of  Agglutination. — Clear  fluid  blood  serum 
to  be  tested  for  specific  agglutinins  is  diluted  with  broth  or  with 
salt  solution  to  make  mixtures  containing  one  part  of  the  serum 
in  5,  10,  20,  40,  80  and  160  parts  of  the  mixture.  This  is  con- 
veniently done  by  means  of  the  Wright  capillary  pipette,  or 
graduated  pipettes  may  be  employed.  To  each  dilution  of  serum 
an  equal  amount  of  a  very  young  (preferably  two  to  six  hours 


212  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

old)  broth  culture,  or  a  suspension  of  an  active  young  agar  cul- 
ture in  broth  or  salt  solution,  is  added.  The  reaction  may  be 
observed  by  mixing  small  quantities  (loopfuls)  on  a  large  cover- 
glass  and  studying  the  mixture  microscopically  as  a  hanging 
drop,  or  by  mixing  larger  quantities  in  small  tubes  and  incubating 
them  at  37°  C.  Control  specimens  free  from  serum  and  contain- 
ing normal  serum  should  be  set  up  at  the  same  time  for  compari- 
son, as  many  bacteria  may  be  agglutinated  somewhat  by  normal 
serum  in  a  dilution  of  one  to  ten,  and  sometimes  the  organisms 
in  the  culture,  especially  if  it  is  too  old,  may  be  already  grouped 
together  somewhat  or  may  spontaneously  clump  during  the  ex- 
periment. Some  practice  is  necessary  before  one  can  estimate 
agglutinins  reliably  and,  on  the  whole,  accuracy  is  more  easily 
attained  with  the  macroscopic  test.  For  agglutination  tests 
requiring  only  moderate  accuracy,  dried  blood  may  be  used, 
the  dilutions  being  prepared  by  comparison  of  colors  with  an 
empirical  standard. 

Bactericidal  Substances,  Alexin. — Nuttall  (1886)  showed 
that  normal  blood  is  capable  of  killing  bacteria  and  that  this 
germicidal  property  is  destroyed  by  heating  the  blood  to  55°  C. 
for  thirty  minutes.  Buchner  confirmed  these  observations  and 
showed  further  that  the  germicidal  property  is  resident  in  the 
serum  and  not  exclusively  in  the  cells  of  the  blood  as  taught  by 
MetchnikofL  To  this  germicidal  substance  Buchner  gave  the 
name  alexin,  and  he  ascribed  the  normal  resistance  to  infection 
exhibited  by  the  healthy  animal,  as  well  as  the  heightened  resist- 
ance of  the  immunized  animal,  to  this  substance.  It  will  have 
been  noted  that,  historically,  these  discoveries  followed  Metch- 
nikofPs  first  observations  on  the  phagocytes,  and  preceded 
the  discovery  of  antitoxins,  agglutinins  and  precipitins,  and 
thus  presented  the  first  proof  of  the  existence  of  soluble  anti- 
infectious  agents.  These  bactericidal  substances  are  now  con- 
sidered to  be  identical  with  the  bacteriolysins  and  will  be 
considered  with  them  under  the  more  general  heading  of 
cytolysins. 


REACTION    OF    THE    HOST    TO    INFECTION  213 

Cytolysins. — Pfeiffer  (1896)  found  that  guinea-pigs,  when 
injected  repeatedly  with  non-fatal  doses  of  cholera  germs,  reacted 
to  this  treatment  by  producing  a  something  which  would  dissolve 
these  bacteria.  This  new  property  was  present  in  the  blood  and 
also  in  the  peritoneal  fluid.  The  substance  was  called  bacterioly- 
sin.  Subsequent  investigators  have  shown  that  bacteriolysins 
can  be  produced  for  a  great  variety  of  micro-organisms,  although 
in  none  can  the  reaction  be  better  demonstrated  than  in  the 
cholera  vibrio  originally  employed  by  Pfeiffer.  Lysins,  or 
dissolving  substances,  have  been  produced  for  very  many  other 
kinds  of  cells  also,  of  which  those  for  red  blood  cells  (hemolysins) 
are  perhaps  the  most  important.  It  seems  to  be  possible  to 
produce  a  lysin  (cytolysin)  for  any  kind  of  cells  by  injecting  these 
cells  into  an  appropriate  animal. 

Cytolysins,  including  bacteriolysins,  are  active  only  when 
comparatively  fresh.  Upon  standing  for  a  day  at  room  tem- 
perature, or  upon  heating  to  56°  C.  for  30  minutes,  the  cytolytic 
power  disappears.  This  power  is,  however,  restored  in  a  re- 
markable manner  if  the  cytolysin  and  the  cells  to  be  dissolved  are 
injected  together  into  a  normal  animal,  for  example  into  the 
peritoneal  cavity  of  a  guinea-pig,  or  if  a  fresh  normal  blood  serum 
be  added  to  the  mixture  in  the  test-tube.  The  experiment  results 
as  follows: 

Immune  serum  +  cholera  germs  =  Bacteriolysis. 

Immune  serum  (old  or  heated)  -f  cholera  germs  =  No  bacteriolysis. 
Normal  serum  +  cholera  germs  =  No  bacteriolysis. 

Immune  serum  (old  or  heated)  -(-  normal  serum + cholera  germs 

=  Bacteriolysis. 

This  experiment  proves  that  the  cytolytic  property  of  the  serum 
depends  upon  the  presence  of  at  least  two  recognizably  different 
substances,  one  of  which  is  present  in  fresh  normal  serum  and 
in  fresh  immune  serum  but  is  destroyed  on  standing  or  by  heating, 
and  a  second  which  is  present  in  the  immune  serum  and  which 
is  not  destroyed  so  readily. 


214 


GENERAL  BIOLOGY    OF   MICRO-ORGANISMS 


Ehrlich  explains  the  formation  of  cytolysins  by  the  same 
kind  of  reasoning  as  was  applied  to  antitoxins  and  precipitins. 
The  resulting  side-chain  would  be  considered  of  the  same  sort 
as  in  the  latter  class  of  substances,  that  is  a  receptor  of  the 
second  order  with  a  haptophorous  group  by  which  to  combine 
with  the  foreign  cell,  and  a  zymophorous  group  to  bring  about 
its  solution,  were  it  not  for  the  observed  facts  given  in  the  experi- 
ment outlined  above,  which  demonstrate  the  presence  of  two 
distinct  substances  in  the  cytolytic  complex.  A  new  picture  is 
here  necessary  and  it  is  furnished  by  making  a  joint  in  the  arm 


FIG.  87. — Receptors  of  the  third  order.  (Journ.  A.  M.  A  ,  1905,  J.  1369.)  c. 
Cell  receptor  of  the  third  order — an  amboceptor;  c,  one  of  the  haptophores  of  the 
amboceptor  with  which  the  foreign  body,  /,  (antigen)  may  unite;  g,  the  other 
haptophore  of  the  amboceptor  with  which  complement,  k,  may  unite;  /?, 
haptophore  of  the  complement;  z,  zymophore  of  the  complement. 

of  the  receptor  of  the  second  order  in  which  the  fermentative 
property  is  supposed  to  reside,  separating  off  the  zymophorous 
group  as  a  separate  substance  and  leaving  a  branched  figure  with 
two  combining  or  haptophorous  elements,  one  capable  of  com- 
bining with  the  foreign  cell  and  the  other  capable  of  combining 
with  the  cytolytic  ferment  of  normal  serum  and  so  bringing  its 
action  to  bear  upon  that  particular  cell.  The  receptor  of  the 
third  order  is  called,  in  accordance  with  this  conception  of  its 
relationships,  amboceptor,  because  it  acts  as  a  receptor  at  two 


REACTION   OF   THE   HOST   TO   INFECTION 


215 


points.  It  is  also  called  intermediary  body,  immune  body  and 
sensitizer.  The  other  component  of  the  lytic  complex,  which 
is  thermolabile  and  which  is  present  in  normal  serum,  is  called 
complement  or  cytase,  and  by  some  authors  (Bordet)  alexin.1 
It  will  be  noted  that  only  a  part  of  the  cytolysin  is  produced 
by  the  body  in  its  reaction  to  invasion,  namely,  the  immune  body. 
Deviation  of  Complement. — Neisser  and  Wechsberg  observed 
that  the  bactericidal  power  of  a  given  immune  serum  (bacteriolytic 
amboceptor),  when  combined  with  a  constant  amount  of  normal 
serum  (complement)  and  a  constant  amount  of  a  bacterial  sus- 
pension (antigen),  increased  progressively  with  progressive 
dilution  of  the  immune  serum  to  a  certain  point,  after  which  it 
diminished  again.  The  following  data  taken  from  Citron  illus- 
trate the  experiment: 


Typhoid  culture  (antigen) 

Immune  serum 
(amboceptor) 

Fresh   serum 
i  :  12 
(complement) 

Colonies  produced 
by  plating   after 
3  hrs.  at  37°  C. 

0.5  c.c.  1/5000  
0.5  c.c.  1/5000  
0.5  c.c.  1/5000  
0.5  c.c.  1/5000  
0.5  c.c.  1/5000  

I/IOO  C.C. 

1/5000  c.c. 

I/2OOOO  C.C. 

1/30000  c.c. 
1/50000  c.c. 

0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
o.  5  c.c. 

Many  thousand 
Many  thousand 
200 
o 
60 

o.  5  c.c.  1/5000  

I/2OOOOO  C.C. 

o.  5  c.c. 

Many  thousand 

Neisser  and  Wechsberg  have  undertaken  to  explain  this 
result  by  supposing  that  the  excessive  number  of  amboceptors 
present  in  the  more  concentrated  solutions  of  immune  serum 
hinders  cytolysis  because  some  of  them  combine  with  the  antigen 
by  means  of  their  cytophile  groups  while  others  are  combining 
with  the  complement  by  means  of  their  complementophile 
groups,  and  as  a  result  the  mixture  contains  combinations  of 
amboceptor  with  antigen,  and  of  amboceptor  with  complement, 
but  practically  no  combinations  of  the  three  elements  together. 
There  are  grave  reasons  for  questioning  the  accuracy  of  this 

1  This  use  of  the  term  alexin  would  seem  to  be  undesirable,  for  Buchner  employed 
the  term  to  designate  the  whole  bactericidal  or  cytolytic  complex  before  the  possi- 
bility of  recognizing  two  separate  elements  was  clearly  recognized. 


2l6 


GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 


assumption,  as  it  has  been  shown  by  Bordet  that  amboceptor 
does  not  unite  with  complement  in  the  absence  of  antigen.  It 
seems  more  probable  that  some  other  factor,  such  perhaps  as  a 
marked  agglutination  of  the  bacteria  in  the  stronger  solutions, 
may  serve  to  protect  them  from  the  bacteriolytic  action. 

Fixation  of  Complement.- — As  has  been  mentioned,  it  is  pos- 
sible to  produce  cytolysins  for  red  blood  cells.  This  is  commonly 
done  by  injecting  the  washed  blood  corpuscles  of  a  sheep  (o.i  c.c. 
+0.5  c.c.  salt  solution)  into  a  rabbit  intravenously  three  or  four 
times  at  intervals  of  five  days.  The  serum  of  the  rabbit  becomes 


FIG.  88.- 


•Illustrating  the  conception  of  deviation  of  complement. 
b,  antigen;  k,  complement. 


a,  Amboceptor; 


strongly  hemolytic  for  sheep's  cells.  The  blood  is  drawn  from 
the  carotid  artery,  the  serum  separated,  rendered  perfectly 
clear  and  after  heating  to  56°  C.  for  30  minutes  is  stored  in  hermet- 
ically sealed  ampoules  containing  i  c.c.  each,  in  a  low  tempera- 
ture refrigerator.  When  this  hemolytic  amboceptor  is  diluted  to 
the  proper  point,  which  must  be  ascertained  by  trial  and  error, 
it  will  just  cause  the  complete  hemolysis  of  a  definite  quantity 
of  washed  sheep's  corpuscles  (0.2  c.c.  of  a  5  per  cent  suspension) 
when  combined  with  o.i  c.c.  of  a  10  per  cent  solution  of  fresh 
normal  serum  of  a  guinea-pig  (complement).  The  mixture  of 
this  quantity  of  the  immune  serum,  which  may  now  be  called 
one  unit  of  hemolytic  amboceptor,  with  0.2  c.c.  of  freshly  prepared 
5  per  cent  suspension  of  washed  sheep's  corpuscles  produces  a 


REACTION   OF    THE   HOST   TO   INFECTION  217 

reagent  which  serves  for  the  detection  of  complement  and  the 
approximate  estimation  of  its  amount  in  an  unknown  mixture. 
By  the  use  of  such  a  reagent  it  is  possible  to  show  that  complement 
is  destroyed  or  used  up  in  various  specific  cytolytic,  proteolytic. 
and  precipitin  reactions.  Thus  Bordet  and  Gengou  mixed 
together  typhoid  bacilli  (antigen),  heated  typhoid-immune 
serum  (amboceptor)  and  fresh  normal  serum  (complement) 
and  incubated  the  mixture.  After  an  hour  the  hemolytic  ambo- 
ceptor and  sheep's  blood  cells  were  added  and  incubation  con- 
tinued. No  hemolysis  resulted,  showing  that  the  complement 
added  in  the  first  place  had  been  used  up,  "fixed,"  as  a  result 
of  a  reaction  with  the  typhoid  bacilli  and  typhoid  amboceptor. 
This  is  the  phenomenon  of  fixation  of  complement.  Obviously 
it  lends  itself  to  use  as  a  test  for  the  presence  of  a  specific 
antigen  or  for  the  presence  of  specific  amboceptor.  Its  more 
definite  application  will  require  subsequent  mention. 

Opsonins. — Wright  and  Douglas  (1903)  showed  that  blood 
serum  contains  a  something  which  affects  bacterial  cells,  soaked 
in  the  serum,  in  such  a  way  that  they  are  more  readily  ingested 
by  the  living  leukocytes.  To  this  substance  they  gave  the 
name  "opsonin"  (opsono,  I  prepare  victuals  for).  Substances 
of  this  sort  are  present  in  normal  blood,  but  are  increased  as  a 
reaction  following  infection.  It  would  seem  that  more  than 
one  substance  may  act  upon  bacterial  cells  in  this  manner,  for 
Neufeld  has  shown  that  the  opsonic  power  of  normal  serum  may 
be  destroyed  by  heating  to  56°  C.,  while  the  similar  property  of 
immune  serum  remains  after  this  treatment.  It  is  not  yet  con- 
clusively proven  that  opsonins  are  separate  substances  entirety 
distinct  from  bacteriolysins  and  agglutinins,  but  it  has  been  shown 
that  opsonic  power  of  a  serum  does  not  correspond  to  its  con- 
centration to  that  of  the  other  antibodies,  and  some  other 
element  must,  therefore,  be  a  factor.  Hektoen  considers  the 
opsonins  to  be  distinct  bodies,  different  from  lysins  and  agglutin- 
ins. The  study  of  opsonins  has  done  much  to  bring  about 
harmony  between  the  followers  of  Metchnikoff,  with  their  tendency 


2l8  GENERAL  BIOLOGY   OF  MICRO-ORGANISMS 

to  emphasize  the  importance  of  phagocytosis,  and  the  followers 
of  Buchner  and  Ehrlich,  who  fixed  their  attention  largely  upon 
the  substances  dissolved  in  the  body  fluids. 

Anti-aggressins,  Specific  Proteolysins. — Various  substances 
produced  in  the  body  as  a  result  of  infection  show  particular 
ability  to  combat  the  effects  of  the  soluble  products  of  the  para- 
site to  which  the  name  aggressins  has  been  given  (see  page  205). 
Knowledge  of  these  substances  and  their  behavior  is  still  some- 
what incomplete,  but  they  seem  to  be  particularly  concerned 
with  the  parental  digestion  of  foreign  proteins,  a  process  in  which 
cystolysis  may  be  regarded  as  a  beginning  stage.  Whereas, 
however,  cytolysis  is  concerned  with  the  disintegration  of  formed 
material,  these  substances  now  under  consideration  act  particularly 
upon  proteins  already  in  solution.  In  many  instances  the  products 
of  the  first  stages  in  this  parental  digestion  are  toxic  (disintegra- 
tion of  tuberculin  and  of  egg-white),  and  some  of  the  symptoms 
of  infectious  disease,  such  as  fever,  have  been  ascribed  to  them. 
In  their  general  characters  these  lytic  substances  are  wholly 
analogous  to  the  cytolysins  and  their  action  is  due  to  at  least  two 
components,  an  amboceptor  and  a  complement. 

Source  and  Distribution  of  Antibodies. — The  exact  source 
of  the  antibodies  dissolved  in  the  body  fluids  is  unknown.  All 
agree  that  they  are  derived  from  cells.  Metchnikoff  regards 
the  phagocytic  cells  as  the  important  source;  Ehrlich  does  not 
specify,  but  it  would  seem,  in  accordance  with  his  theory,  that 
any  cell  capable  of  being  affected  by  the  foreign  substance  should 
be  capable  of  throwing  off  cell  receptors  (antibodies)  to  combine 
with  it.  Many  investigators  consider  antibody  formation  to  be 
a  common  property  of  many  kinds  of  cells,  but  more  especially 
of  relatively  undifferentiated  cells  such  as  those  of  the  connective 
tissue. 

Antibodies  are  present  in  greatest  concentration  in  the  blood 
and  lymph.  They  are  absent  or  present  only  in  small  amount 
in  the  serous  fluids  of  the  pleural,  pericardial,  peritoneal  and 


REACTION    OF    THE   HOST   TO    INFECTION  2IQ 

joint  cavities,  and  in  the  cerebrospinal  fluid.1  Parasites  in 
these  locations  are  less  readily  influenced  by  antibodies  circulating 
in  the  blood,  so  that  localized  infections  may  continue  in  these 
regions  in  spite  of  a  considerable  concentration  of  antibodies  in 
the  body  generally. 

Allergy. — Allergy  is  a  term  invented  by  Von  Pirquet  to 
designate  the  condition  of  altered  reactivity  on  the  part  of  the 
body  which  comes  about  as  a  result  of  infection.  A  few  of  the 
phenomena  which  may  be  included  under  this  term  have  been 
considered  above  in  this  chapter.  Many  of  these  alterations  in 
bodily  function  are  manifestly  of  advantage  to  the  host  in  limiting 
the  activities  of  the  parasite,  neutralizing  its  poisonous  products, 
and  even  in  destroying  and  removing  the  parasite  itself.  Some  of 
them,  such  as  specific  precipitation,  seem  to  serve  no  important 
purpose,  while  others,  such  as  cytolysis  and  proteolysis  actually 
lead  sometimes  to  results  very  harmful  to  the  host,  although 
their  usual  effect  is  favorable.  Many  of  the  recognized  weapons 
which  the  body  employs  in  its  battle  against  parasites  are  still 
imperfectly  understood,  and  there  are  doubtless  many  factors 
involved  in  this  relation  which  are  not  yet  capable  of  definite 
recognition.  Of  those  agents  mentioned  above,  the  phagocytes 
are  ready  for  immediate  defense  as  soon  as  the  body  is  invaded 
by  the  parasite.  Hyperplasia  and  encapsulation  require  more 
time,  probably  one  to  four  weeks.  The  chemical  antibodies, 
antitoxins,  agglutinins,  cytolysins  and  opsonins,  although  possibly 
present  in  small  amounts  in  the  normal  body  fluids,  become 
definitely  increased  in  from  eight  to  twelve  days  after  the  entrance 
of  the  parasite,  an  interval  approximately  equal  to  the  incubation 
period  of  some  infectious  diseases.  These  various  agents  have 
much  to  do  in  determining  the  manifestations  and  course  of  the 
disease  as  well  as  the  final  outcome,  and  as  we  shall  see,  they  also 
play  a  part  in  immunity. 

1  See  Flexner,  Harbin  Lectures,  Journ.  of  the  State  Medicine,  March,  April,  May 
1912. 


CHAPTER  XIII. 

IMMUNITY  AND  HYPERSUSCEPTIBILITY.     THEORIES 
OF  IMMUNITY. 

Immunity. — Immunity  is  that  condition  of  a  living  organism 
which  enables  it  to  escape  without  contracting  a  disease  when 
fully  exposed  to  conditions  which  normally  give  rise  to  that  disease. 
Immunity  may  depend  upon  many  different  factors,  or  upon 
only  one  of  a  great  variety.  In  general,  we  shall  find  that  it 
depends  very  largely  upon  those  factors  which  we  have  already 
considered  in  the  preceding  chapters,  such  as  the  possession  of 
anatomical  structures  or  habits  of  life  which  render  invasion  by 
the  particular  parasite  impossible,  or  the  possession  of  a  body 
structure,  physically  or  chemically  not  adapted  for  the  growth 
of  the  particular  disease  virus,  or  the  ability  to  harbor  the  particular 
parasite  as  a  commensal  without  suffering  injury,  or  the  ability  to 
react  against  the  invading  parasite  and  destroy  it  by  phagocytosis 
or  by  cytolysis,  neutralize  its  poisons  by  antitoxins,  or  limit  its 
activity  by  encapsulation.  Immunity  is  ordinarily  considered 
under  two  heads,  Natural  Immunity,  or  that  present  as  a  part  of 
the  individual's  birthright,  and  Acquired  Immunity,  that  which 
follows  as  the  result  of  some  experience  of  the  individual. 

Immunity  of  Species. — Natural  immunity  to  certain  diseases 
is  possessed  by  certain  species  of  animals.  Where  the  morphology 
and  physiology  is  quite  different  from  that  of  the  usual  victims 
of  the  disease,  immunity  might  be  expected.  Thus  cold-blooded 
vertebrates,  fish,  amphibians  and  reptiles,  are  immune  to  many 
diseases  of  mammals,  apparently  because  of  the  different  tem- 
perature of  their  tissues.  In  other  instances  the  difference 
in  resistance  between  two  species  of  animals  seems  to  be  correlated 

220 


IMMUNITY    AND    HYPERSUSCEPTIBILITY  221 

with  difference  in  food  habits.  Thus  the  carnivorous  mammals 
are  relatively  insusceptible  to  anthrax  and  tuberculosis,  diseases 
natural  to  the  herbivora.  Many  infectious  diseases  of  man 
are  not  readily  transmissible  to  animals,  for  example,  typhoid 
fever,  syphilis,  pneumonia,  and  in  some  instances  it  has  so  far 
been  impossible  to  infect  animals,  as  for  example  with  scarlet 
fever  and  gonorrhea.1 

Racial  Immunity. — Within  a  species  there  is  moreover  a 
racial  difference  in  resistance  to  natural  infection.  Thus  the 
pure-bred  dairy  cattle  are  more  susceptible  to  tuberculosis  than 
other  cattle,  and  Yorkshire  swine  are  relatively  less  susceptible 
to  swine  erysipelas.  In  man,  the  relation  of  race  to  susceptibility 
is  not  very  clear.  The  examples  of  supposed  racial  immunity 
have  not  proved  to  be  so  definite  as  had  been  assumed  at  first. 
Thus  the  supposed  immunity  of  African  natives  to  syphilis  has 
vanished  with  their  increasing  contact  with  civilization  and 
with  this  accompanying  disease.  In  the  case  of  malaria  the 
supposed  racial  immunity  of  negroes  seems  to  be  an  acquired 
immunity  due  to  severe  attacks  of  the  disease  in  childhood. 
There  is,  however,  some  evidence  that  prolonged  contact  with  a 
disease  through  many  generations  may  result  in  a  relative  resist- 
ance, so  that  the  disease  assumes  a  milder  form  in  such  a  race  of 
people — a  sort  of  inherited  acquired  immunity.  Such  considera- 
tions have  been  brought  forward  to  explain  the  relatively  high 
resistance  to  tuberculosis  shown  by  the  Hebrews  as  compared 
with  the  American  Indians. 

Individual  Variations. — Individual  variations  in  resistance 
to  infection  are  commonly  observed.  They  may  depend  in  part 
upon  age,  condition  of  nutrition,  fatigue,  exposure  or  intoxica- 
tion, but  they  are  ascribed  also  to  differences  in  anatomical 
structure  (shape  of  the  thorax  in  tuberculosis).  Individuals 
especially  susceptible  to  a  disea.se  are  said  to  possess  an  idiosyn- 
crasy for  it.  The  physiological  mechanisms  upon  which  varia- 
tions in  individual  resistance  depend  are  not  clearly  understood. 

1  Kolle  und  Wassermann,  II  Auflage,  Bd.  IV,  p.  693  (1912). 


222  GENERAL  BIOLOGY   OF    MICRO-ORGANISMS 

Doubtless,  the  number  and  activity  of  the  white  blood  cells  and 
the  nature  and  amount  of  bactericidal  substances  in  the  blood 
play  a  part  in  some  instances. 

Acquired  Immunity. — Acquired  immunity  results  from  some 
experience  affecting  the  individual,  either  an  infection  which  the 
individual  has  survived  or  some  artificial  procedure  of  immuniza- 
tion. There  are  recognized  two  different  kinds  of  acquired 
immunity,  first,  active  immunity  which  is  due  to  the  activity 
of  the  cells  of  the  individual  immunized,  and  second,  passive 
immunity  which  is  produced  by  introducing  into  the  body  material 
(blood  serum)  from  another  animal,  which  contains  substances 
conferring  at  once  an  immunity  upon  the  new  individual. 

Active  Immunity. — Active  immunity  may  be  acquired  by  an 
attack  of  the  disease.  This  immunity  may  endure  for  a  lifetime 
in  some  instances  (yellow  fever,  small-pox,  scarlet  fever)  or  for 
many  years  (typhoid  fever)  or  it  may  be  very  evanescent 
(erysipelas,  pneumonia,  influenza).  Some  diseases  were  at  one 
time  so  universal  that  few  escaped  them,  and  individuals  used 
to  be  purposely  exposed  or  inoculated  in  order  to  contract  the 
disease  and  gain  the  resulting  immunity.  Inoculation  of  small- 
pox seems  to  have  been  practised  in  China  about  1000  A.  D.  and 
in  India  as  early  as  the  twelfth  century,  and  it  was  introduced  into 
Europe  in  1721  by  Lady  Montague  and  was  employed  very 
extensively  in  Europe  and  America  during  that  century. 

Active  immunity  may  also  be  produced  without  causing  a 
definite  attack  of  the  disease.  This  may  be  accomplished  in  a 
variety  of  ways.  Fully  virulent  micro-organisms  may  be  intro- 
duced into  a  part  of  the  body  unfavorable  to  their  development. 
The  subcutaneous  injection  of  cholera  cultures  according  to  the 
method  of  Ferran  and  Haffkine  has  proven  to  be  practically 
without  danger,  and  results  in  immunity.  The  same  principle 
is  ultilized  in  immunizing  cattle  against  pleuro-pneumonia.1 
Introduction  of  virulent  organisms  in  very  minute  doses  has  been 
employed  to  immunize  against  rabies  (Hogyes  method),  and  against 

1  Kolle  und  Wassermann,  II  Auflage,  Bd.  I,  S.  928  (1912). 


IMMUNITY  AND   HYPERSUSCEPTIBILITY  223 

tuberculosis  by  Webb.  In  most  diseases  these  methods  are  re- 
garded as  too  dangerous  for  extensive  use. 

Living  virus,  altered  in  its  virulence,  was  first  used  by  Edward 
Jenner,  when  he  inoculated  with  cow-pox  (vaccinia)  and  induced 
immunity  to  small-pox.  Cow-pox  is  doubtless  due  to  the  organism 
which  causes  small-pox,  attenuated  by  its  life  in  the  body  of  the  cow. 
Viruses  artificially  cultivable  are  attenuated  by  a  variety  of  pro- 
cedures, and  are  employed  to  induce  immunity.  Pasteur's  vaccine 
for  anthrax,  for  chicken  cholera  and  possibly  the  treatment  of 
rabies  with  dried  spinal  cord,  are  examples  of  the  application  of 
this  principle.  Virus  of  extraordinary  virulence  is  sometimes  in- 
jected after  previous  treatment  with  attenuated  organisms,  in 
order  to  confer  a  higher  degree  of  immunity.  Thus  Pasteur 
employed  the  most  virulent  rabies  virus  obtainable,  virus  fixe,  in 
the  immunization  against  rabies. 

Living  virus,  of  full  virulence,  but  apparently  influenced  in  some 
way  by  the  body  fluid  containing  it,  is  employed  in  immunizing 
against  rinderpest  and  against  Texas  fever.  The  bile  of  an  animal 
dying  of  rinderpest  is  injected  subcutaneously  in  doses  of  10  c.c. 
into  cattle.  Kolle  has  shown  that  the  virus  can  be  separated  from 
such  bile  in  fully  virulent  condition;  so  it  appears  that  some  con- 
stituents of  the  bile  restrain  the  activity  of  the  virus.  In  Texas 
fever,  blood  of  young  animals  containing  relatively  few  of  the 
parisites  is  used  to  inject  new  animals. 

Immunization  by  injection  of  dead  microbic  substance  is  now 
extensively  employed  in  the  prophylaxis  of  cholera,  typhoid  fever 
and  plague.  As  a  result  of  such  injections  there  is  a  marked  in- 
crease in  specific  agglutinins  and  bacteriolysins  in  the  blood.  The 
principle  of  general  immunization  is  also  employed  with  some  suc- 
cess in  the  treatment  of  subacute,  chronic  or  recurrent  local 
infections,  the  production  of  antibodies  and  their  circulation  in 
the  blood  and  lymph  exerting  a  favorable  effect  upon  the  local 
lesions.  The  emulsions  of  dead  bacteria  employed  are  called 
bacterial  vaccines. 

The  soluble  products  of  bacterial  growth  are  injected  into 


224  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

animals  to  immunize  them,  especially  in  the  case  of  diphtheria 
and  tetanus,  the  bacteria  of  which  produce  very  powerful  soluble 
toxins.  As  a  result  of  this  treatment  antitoxins  are  produced  and 
circulate  in  the  blood  of  the  animal. 

Bacterial  extracts,  either  those  contained  in  inflammatory 
exudates,  the  so-called  aggressins  of  Bail,  or  extracts  obtained  by 
soaking  bacteria  in  blood  serum  or  in  distilled  water,  the  so-called 
artificial  aggressins  of  Wassermann  and  Citron,  have  proved  of 
value  in  experimental  immunization  of  animals  against  many  dif- 
ferent bacteria.  It  is  claimed  that  the  reactions  to  injection  are 
exceptionally  mild,  while  the  resulting  immunity  is  more  solid. 
Certain  products  of  the  disintegration  of  typhoid  bacilli  have  been 
obtained  by  Vaughan,  which  possess  considerable  immunizing 
power,  but  apparently  only  slight  toxicity.  None  of  these  bac- 
terial extracts  has  yet  passed  beyond  the  experimental  stage  in  the 
immunization  of  man  against  a  disease. 

A  certain  slight  grade  of  immunity  may  be  secured  in  some 
instances  by  procedures  which  seem  to  bear  no  relation  to  the 
specific  micro-organisms  in  question.  Thus  the  injection  of  cul- 
tures of  B.  prodigiosus  and  B.  pyocyaneus  results  in  an  increased 
resistance  to  infection  with  anthrax.  Similar  increased  resistance 
has  been  observed  to  follow  a  simple  surgical  procedure,  such  as 
section  of  the  sciatic  nerve.  The  explanation  of  these  results  is 
not  clear,  but  perhaps  the  effect  may  be  attributed  to  a  general 
stimulation  of  the  body  defenses,  especially  the  phagocytes. 

Passive  Immunity. — Passive  immunity  is  produced  by  inject- 
ing into  the  body  a  fluid  taken  from  another  animal  which  contains 
antitoxins,  bacteriolysins,  opsonins  or  other  substances  known 
as  immune  bodies.  The  animal  which  furnishes  the  immune 
bodies  must  be  first  actively  immunized,  and  it  possesses  an  ac- 
tive immunity.  If  its  blood  plasma  be  drawn  and  injected  into 
a  child,  the  child  acquires  a  borrowed  immunity  without  the 
necessity  of  any  active  participation  of  its  own  cells  in  the  pro- 
duction of  immune  bodies.  The  possibility  of  producing  such 
passive  immunity  has  been  demonstrated  in  a  number  of  diseases. 


IMMUNITY  AND  HYPE  INSUSCEPTIBILITY  225 

In  some  instances  the  effect  of  the  serum  is  antitoxic  (diphtheria 
and  tetanus),  in  others  it  is  bacteriolytic  (cholera),  while  in 
other  instances  the  nature  of  the  dominant  antibodies  is  not 
definitely  known. 

Combined  Active  and  Passive  Immunity. — Various  procedures 
have  been  devised  to  produce  immunity  by  introducing  at,  or 
nearly  at,  the  same  time  the  infectious  agent  or  its  products  and 
the  serum  of  an  immune  animal  containing  protective  substances. 
The  combination  of  immune  blood  with  virus  of  full  strength  is 
used  in  immunizing  animals  against  rinderpest,  foot-and-mouth 
disease  and  hog  cholera,  all  being  diseases  due  to  filterable 
agents;  and  also  in  immunizing  hogs  against  hog  erysipelas 
(B.  rhusiopathice).  The  combined  injection  of  attenuated 
virus  and  immune  serum  'is  employed  especially  in  Sobernheim's 
method  of  preventive  inoculation  against  anthrax.  Besredka 
has  employed  dead  bacteria  combined  with  their  specific  immune 
serum  in  immunizing  against  typhoid  fever,  plague  and  cholera. 

The  Mechanisms  of  Immunity. — Certain  biological  factors 
in  the  phenomenon  of  immunity  are  now  clearly  recognizable 
and  readily  demonstrable.  The  activity  of  the  phagocytes,  first 
emphasized  by  Metchnikoff  and  believed  by  him  to  be  the  sole 
important  factor  in  the  defense  of  the  body,  is  easily  observed 
in  immunity  to  many  diseases.  The  dependence  of  phagocytic 
activity  upon  dissolved  substances  in  the  body  fluids  (opsonins) 
is  also  demonstrated  beyond  doubt.  Phagocytosis  is,  perhaps, 
the  factor  of  most  general  operation  in  immunity  to  all  sorts  of 
disease.  The  antitoxins  stand  forth  prominently  as  powerful 
factors  in  immunity  to  two  important  diseases,  diphtheria  and 
tetanus,  and  the  bacteriolysins  are  undoubtedly  of  greatest  im- 
portance in  the  case  of  Asiatic  cholera,  and  probably  also  in  ty- 
phoid and  plague.  In  most  instances  the  immunity  seems  to 
depend  upon  several  different  factors,  phagocytosis,  opsonins, 
bacteriolysins,  antitoxins,  and  perhaps  substances  of  unknown 
nature.  In  some  instances  of  immunity  there  is  no  particular 
excess  of  these  immune  bodies  demonstrable  in  the  blood,  and 
15 


226  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

nearly  always  an  immunity  remains  long  after  such  an  excess 
has  disappeared.  It  would  seem  that  the  ability  of  the  cells  of 
the  body  to  respond  promptly  to  invasion  is  often  developed  by 
experience  with  one  such  invasion,  and  that  this  ability  may  re- 
main for  a  long  time  as  a  factor  in  immunity. 

Hypersusceptibility  or  Anaphylaxis. — If  a  guinea-pig  be  in- 
jected with  a  small  amount  of  a  protein,  such  as  egg-albumen  or 
blood  serum  of  the  horse,  and  then  after  an  interval  of  ten  to 
twenty  days  be  injected  with  a  second  dose  of  the  same  protein 
of  good  size  (0.5  to  5  grams),  the  animal  usually  develops  symptoms 
of  nervous  intoxication  and  often  dies  within  a  half  hour.  Inas- 
much as  normal  guinea-pigs  withstand  enormous  doses  of  such 
protein  substances,  it  is  evident  that  the  first  injection  has  brought 
about  some  change  in  the  animal,  an  altered  reactivity,  which  re- 
sults in  the  intoxication  after  the  second  dose.  That  this  phe- 
nomenon of  hypersusceptibility  or  anaphylaxis  (  =  against  pro- 
tection) bears  a  definite  relation  to  immunity  may  be  illustrated 
by  an  experiment  in  which  typhoid  bacilli  are  substituted  for 
the  soluble  protein.  If  a  guinea-pig  be  immunized  by  repeated 
doses  of  the  killed  micro-organisms  he  is  able  to  resist  inoculation 
with  an  ordinarily  fatal  dose  of  the  living  germs,  which  are 
quickly  killed  and  dissolved  by  the  specific  bacteriolysins  in  the  body 
fluids.  However,  if  such  an  immune  guinea-pig  be  injected  with 
a  proper  dose  of  dead  organisms,  which  would  not  kill  a  normal 
animal,  he  may  quickly  succumb.  The  ability  of  the  body  fluids 
of  the  immune  animal  to  disintegrate  the  bacterial  cells  rapidly 
would  seem  to  be  the  factor  upon  which  depends  not  only  its 
immunity  to  the  small  dose  of  living  germs,  but  also  its  exagger- 
ated sensitiveness  to  dead  germ  substance.  The  products  of  the 
rapid  parenteral  digestion  of  the  foreign  protein  would  seem  to  be 
the  cause  of  the  symptoms  of  intoxication.  The  essential  unity 
of  the  substances  upon  which  immunity  and  anaphylaxis  depend 
has  been  emphasized  by  Von  Pirquet1  and  his  co-workers. 

1  Von  Pirquet:  Allergy.  Archives  of  Internal  Medicine,  1911,  Vol.  VII,  pp.  259-288 ; 
pp. 383-436. 


IMMUNITY   AND   HYPERSUSCEPTIBILITY  227 

Theories  of  Immunity. — Early  theories  of  immunity  were 
based  upon  meager  observations.  The  idea  that  an  attack  of  a, 
disease  left  behind  in  the  body  something  which  prevented  the 
subsequent  entrance  of  that  disease  was  formulated  by  Chauveau 
in  1880  as  the  so-called  retention  hypothesis.  In  the  same  year 
Pasteur  expressed  the  idea  that  an  attack  of  a  disease  removed 
something  from  the  body  and  so  exhausted  the  soil  as  far  as  that 
particular  disease  was  concerned.  Neither  of  these  ideas  was 
new  at  that  time,  and  neither  of  them  pretended  to  any  very 
definite  or  specific  application  to  phenomena  observed  in  immu- 
ity,  but  only  to  the  general  phenomenon  of  immunity  itself. 
The  discovery  of  phagocytosis  by  Metchnikoff  in  1884  was  the 
first  observation  of  a  definite  phenomenon  which  appeared  to 
explain  the  facts  of  immunity.  The  phagocytic  theory,  which 
grew  out  of  this  observation,  was  an  attempt  to  ascribe  immunity 
in  general  to  this  one  phenomenon  of  phagocytosis.  With  the 
observation  of  the  bactericidal  substances  in  solution  in  the  blood 
plasma  by  Nuttall  and  by  Buchner,  of  the  antitoxins  by  von 
Behring  and  the  bacteriolysins  by  Pfeiffer,  there  developed  at- 
tempts to  ascribe  all  the  observed  facts  of  immunity  to  these 
factors,  resulting  in  the  alexin  theory  and  the  antitoxin  theory 
of  immunity.  More  intimate  study  of  the  dissolved  immune 
bodies  lead  to  the  formulation  of  a  hypothesis  to  explain  their 
formation,  composition  and  action,  the  side-chain  theory  of  Ehr- 
lich,  which  has  been  of  great  value  as  a  working  hypothesis  and 
as  a  central  conception  about  which  to  arrange  the  observed  facts 
relating  to  these  dissolved  substances.  The  elementary  concepts 
of  this  theory  have  been  given  in  the  preceding  chapter. 

In  brief,  Ehrlich  pictures  the  living  cell  as  a  chemical  unit 
possessing  numerous  and  varied  combining  groups  or  side-chains 
capable  of  uniting  with  substances  in  contact  with  the  cell. 
The  toxin  molecule  is  conceived  as  a  substance  containing  at 
least  two  distinct  chemical  groups,  one  which  serves  for  attach- 
ment to  the  side-chain  of  the  cell  and  the  other  serving  to  bear 
the  poisonous  properties.  The  union  of  the  toxin  with  the  cell 


228  GENERAL  BIOLOGY   OF   MICRO-ORGANISMS 

results  in  destruction  of  the  side-chains  attacked,  and  in  regen- 
erating these  the  cell  over-compensates,  the  excess  side-chains, 
receptors  of  the  first  order  (see  page  209),  being  set  free  into  the 
blood  and  constituting  the  antitoxin,  which  is  capable  of  neutral- 
izing l  toxin  there  or  in  the  test-tube.  The  assumption  of  two 
chemical  groups  in  the  toxin  molecule  is  strengthened  by  the 
observation  that  diphtheria  toxin  changes  on  standing  so  that 
its  poisonous  property  is  much  diminished  without  corresponding 
loss  of  ability  to  combine  with  antitoxin.  Such  changed  toxin, 
in  which  the  haptophorous  group  persists  while  the  toxophorous 
group  has  degenerated,  is  called  toxoid.  In  order  to  explain  the 
formation  and  structure  of  agglutinins  and  precipitins,  Ehrlich 
assigned  a  more  complex  composition  to  the  side-chains  which 
constitute  these  substances,  leading  to  the  conception  of  a  receptor 
of  the  second  order  (see  page  210),  with  its  haptophorous  and 
zymophorous  groups.  In  the  case  of  the  cytolysins,  a  further 
amplification  of  the  idea  was  necessary  to  explain  the  observed  fact 
that  the  cytolysis  is  due  to  two  components,  one  of  which  is  a 
thermolabile,  normal  constituent  of  the  blood  and  not  increased 
as  a  result  of  immunization,  the  other  being  a  thermostable  sub- 
stance which  is  produced  as  a  result  of  the  immunization  process. 
This  latter  immune  body,  the  receptor  of  the  third  order,  was  there- 
fore pictured  as  a  double  receptor  (amboceptor)  capable  of  attach- 
ing on  the  one  hand  the  foreign  body  (antigen)  and  on  the  other 
the  normal  component  necessary  to  complete  the  lytic  complex, 
to  which  component  .the  name  complement  was  given. 

With  the  recognition  of  opsonins  by  A.  E.  Wright  in  1903, 
the  opposing  theories  of  the  French  and  the  German  schools  be- 
gan to  be  reconciled,  and  the  relatively  simple  and  largely  hypo- 
thetical theories  of ^jfnmunity  be^ga'n  to  give  way  to  a  more  exact 
and  necessarily' ••more  complex  science  df  immunology.  Bordet 
and  his -pupils  Reserve  credit  for  leading  the  reaction  against  too 
slavish  adherence  to  theory  in  the  study  of  immunity.  Our 
modern  ideas  are  no  longer  confined  within  the  scope  of  any  one 
theory  and  it  is  necessary  to  recognize  the  existence  of  a  great 


IMMUNITY   AND   HYPERSUSCEPTIBILITY  22Q 

variety  of  phenomena  in  the  interaction  of  the  host  cells  and  their 
secretions  on  the  one  hand  with  the  parasites  and  their  chemical 
products  on  the  other.  The  elementary  conceptions  of  immun- 
ology and  the  primary  language  of  the  science  are  derived  from 
the  old  theories,  especially  from  Ehrlich's  theory,  and  these  theo- 
ries are  an  essential  part  of  the  introduction  to  immunology.1 

1  For  a  concise  presentation  in  English  of  facts  and  practical  experiments  re- 
lating to  immunity,  the  student  is  referred  to  Citron,  Immunity,  translated  by 
Garbat,  Philadelphia,  1912. 


COMPLIMENTS 
OF 


•    :>'.  .     V 

K) 


PART  III. 
SPECIFIC  MICRO-ORGANISMS 


CHAPTER  XIV. 

THE  MOLDS  AND  YEASTS  AND  DISEASES  CAUSED 

BY  THEM. 

The  general  characters  of  molds  and  yeasts  have  been  men- 
tioned in  a  previous  chapter.  The  generic  and  specific  relation- 
ships of  many  of  those  commonly  met  with  by  the  pathological 
bacteriologist  are  in  a  state  of  confusion.  No  claim  of  systematic 
arrangement  is  made  for  the  material  here  presented. 

Mucor  Mucedo. — This  is  the  most  common  species  of  mucor, 
especially  about  barns  and  on  manure.  It  produces  a  network 
of  threads  (mycelium)  in  the  substratum,  and  zygospores  are  pro- 
duced here  by  the  union  of  two  cells.  The  aerial  hyphae  are 
vertical,  about  10  cm.  in  length  and  bear  a  spherical  spore  sac 
(sporangium)  at  the  end.  The  sporangium  is  at  first  yellow, 
later  brown  and  finally  black  and  covered  with  crystals.  The 
contained  spores  are  4  to  6^  wide  by  7  to  ID/*  long.  It  is 
saprophytic. 

Mucor  Corymbifer. — Lichtheim  found  this  mold  growing  on 
a  bread-infusion  gelatin  as  an  accidental  contamination.  The 
growth  is  at  first  white  and  later  gray.  The  spore-bearing  hyphae 
are  long  and  irregularly  branched,  and  each  branch  bears  a  pear- 
shaped  sporangium  10  to  yo/*  in  diameter.  The  contained  spores 
are  small  (2X3^).  Intravenous  injection  of  the  spores  into  rab- 
bits causes  severe  nephritis  and  death  in  two  or  three  days. 

231 


232 


SPECIFIC  MICRO-ORGANISMS 


FIG.  89. — Mucor  mucedo.  i,  A  sporangium  in  optical  longitudinal  section: 
c,  columella;  m,  wall  of  sporangium;  sp,  spores.  2,  A  ruptured  sporangium  with  only 
the  columella  (c)  and  a  small  portion  of  the  wall  (m)  remaining.  3,  Two  smaller 
sporangia  with  only  a  few  spores  and  no  columella.  4,  Germinating  spores.  5, 
ruptured  sporangium  of  Mucor  mucilaginus  with  deliquescing  wall  (m)  and  swollen 
interstitial  substance  (z);  .>•/>,  spores.  (From  Jordan  after  Brefeld.) 


FIG.  90. — Mucor  corymbifer.     (From  Plant  after  Lichthelm.} 


MOLDS    AND    YEASTS   AND    DISEASES    CAUSED   BY   THEM 


233 


The  mold  has  been  found  growing  as  a  parasite  in  the  auditory 
canal. 

More  than  a  hundred  species  of  Mucor  have  been  described 
and  several  of  them  cause  disease  and  death  when  injected  into 
animals. 

Aspergillus  Glaucus.— This  is  very  widely  distributed  in 
nature,  occurring  on  fruits,  moist  bread  and  other  food  substances 
and  very  frequently  as  a  con- 
tamination in  laboratory  cul- 
tures. The  aerial  spore-bear- 
ing hypha  (conidiophore)  is 
erect,  about  i  mm.  long,  swollen 
at  the  end  to  a  diameter  of  20 
to  40;*.  On  the  surface  of  this 
spherical  head  are  numerous 
closely  packed  spore-bearing 
sterigmae,  each  of  which  bears 
at  its  tip  a  chain  of  spherical 
spores  (conidia)  which  are 
budded  off  from  it.  The  coni- 
dia are  gray  to  olive  green  in 
color.  Ascospores  are  also  produced,  grouped  together  as  yellow 
masses,  called  perithecia,  on  the  surface  of  the  medium.  The 
mold  is  not  pathogenic.  Probably  a  considerable  number  of 
different  species  have  been  included  under  this  name. 

Aspergillus  Fumigatus. — The  growth  of  this  mold  is  at  first 
bluish  and  later  grayish-green.  It  is  widely  distributed.  The 
sterigmae  are  unbranched,  thickly  set  on  the  swollen  end  of  the 
spore-bearing  hypha.  The  conidia  measure  2.5  to  3^.  The  for- 
mation of  ascospores  has  also  been  observed.  Aspergillus  fumi- 
gatus  plays  a  part  in  the  heating  of  hay  and  sprouting  barley, 
and  is  the  most  common  of  the  pathogenic  aspergilli.  It  infects 
doves  and  other  birds  naturally,  sometimes  causing  veritable 
epidemics,  and  the  disease  has  been  observed  in  bird  fanciers, 
in  whom  it  runs  a  clinical  course  very  similar  to  that  of  pulmonary 


FIG.  91. — Aspergillus  fumigatus  from  the 
lung  of  a  parrot.     (After  PlauL} 


234 


SPECIFIC   MICRO-ORGANISMS 


tuberculosis.  Fragments  of  the  mycelium  are  found  in  the  spu- 
tum. Doubtless  the  human  disease  is  contracted  from  the  birds 
in  these  cases.  This  mold  has  been  found  as  the  apparent  cause 
of  inflammation  in  the  auditory  canal  in  a  large  number  of  cases 

and  in  the  nasal  fossae  in  a  few  in- 
stances. Various  other  mammals  are 
susceptible  to  inoculation  and  natural 
infection  has  been  observed  in  horses, 
cattle,  sheep  and  dogs. 

Many  other  species  of  pathogenic 
aspergilli  have  been  described,  of  less 
frequent  occurrence  than  A.fumigatus. 
Penicillium  crustaceum  (glaucum) 
is  the  commonest  contaminating  mi- 
cro-organism met  with  in  the  labora- 
tory, and  is  probably  the  most  widely 
distributed  mold.  Ascospores,  similar 
to  those  of  Aspergillus  glaucus  have 
been  observed,  but  they  are  rarely 
produced.  The  aerial  fruiting  hypha 
(conidiophore)  is  erect,  septate  and 
branched  at  the  upper  end  like  a  brush. 
At  the  end  of  these  branches  are  bot- 

FIG.  92. — Pemcillmm   crusta- 
ceum.   Conidiophore^ with  verti-  tie-shaped    stergmae   from  which    the 

conidia     are     constricted     off.     The 


comdia.     XS4C.     (From    Jordan   growth    IS    at  first  white  and   then   It 
after  Strasburger.) 

becomes  blue-green,  the  development 

of  color  beginning  at  the  center.  Penicillium  crustaceum,  or  at 
any  rate  a  certain  variety  of  it,  is  an  important  agent  in  the 
ripening  of  Rocquefort  cheese.  It  is  not  pathogenic,  but  the 
extracts  from  cultures  of  some  varieties  are  poisonous  when  in- 
jected into  animals.  It  is  possible  that  several  distinct  species 
have  been  included  under  this  one  name  of  Penicillium  crustaceum. 
Claviceps  Purpurea. — This  is  a  fungus  parasitic  upon  rye 
and  a  few  other  plants.  The  spores  gain  access  to  the  flower  of 


MOLDS    AND    YEASTS    AND    DISEASES    CAUSED    BY    THEM      235 

rye  and  develop  a  mycelial  mass  which  grows  in  the  utricle,  dis- 
placing the  grain,  the  rudiment  of  which  lies  above  the  mass  of 
the  mold.  Closely  packed  conidiophores  produce  oval  conidra 
and  at  the  same  time  secrete  a  sweet  milky  fluid  which  attracts 
insects  and  thus  furthers  the  distribution  of  the  parasite.  Later 
the  mycelial  mass  produces  sclerotia,  which  are  masses  of  thick- 
walled  cells  containing  starch  and  oil  together  with  specific  poi- 
sonous substances,  and  the  whole  becomes  dry  and  hard  with  black 
outer  covering,  forming  the  ergot  grain,  which  is  considerably 
larger  than  the  normal  rye  grain.  In  autumn  this  falls  to  the 
ground  and  remains  until  spring,  when  numerous  red  stalks  grow 
out  of  it.  Upon  the  swollen  ends  of  these  stalks,  abundant  as- 
cospores  are  produced,  and  these  serve  to  infect  again  the  flowers 
of  the  new  crop  of  rye. 

This  fungus  is  of  great  importance  as  the  source  of  the  drug, 
ergot,  and  as  a  cause  of  food  poisoning,  ergotism,  in  certain  coun- 
tries. It  is  one  example  of  a  mold  parasitic  upon  higher  plants. 
There  are  very  many  different  species  of  such  parasitic  fungi, 
and  they  are  probably  the  best  known  microbic  agents  causing 
diseases  of  plants.1 

Botrytis  Bassiana. — This  mold  was  shown  to  be  the  cause  of 
muscardine,  a  disease  of  silkworms,  by  Bassis  and  Audouin  in 
1837,  a  discovery  following  closely  the  recognition  of  the  itch 
mite,  Sar copies  scabei,  as  the  cause  of  scabies  in  1834.  The  in- 
fected silkworm  becomes  sluggish  and  dies,  and  the  aerial  hyphae 
of  the  fungus  grow  out  from  its  surface  and  pinch  off  round  or 
pear-shaped  conidia.  These  spores  gain  the  surface  of  other 
silkworms  or  butterflies  by  contact  or  by  air  transmission,  and 
germinate,  sending  threads  into  their  bodies.  Sickle-shaped 
spores  are  produced  from  these  inside  the  body,  and  these  grow 
out  into,  threads,  forming  a  mycelial  network  throughout  the 
body  of  the  victim  and  causing  its  death.  It  is  possible  that 

1  For  a  consideration  of  molds  in  relation  to  plant  pathology,  see  Massee, 
Diseases  of  cultivated  plants  and  trees,  New  York,  1910. 


236 


SPECIFIC   MICRO-ORGANISMS 


several  different   species   of   molds   may   be    concerned   in   the 
causation  of  muscardine. 

The  fungus  is  of  interest  because  it  was  probably  the  first 
mold  to  be  recognized  as  a  cause  of  disease,  and  also  because 
it  is  an  example  of  a  large  group  of  fungi  which  attack  various 
insects.  The  disease  muscardine  is,  moreover,  one  of  con- 
siderable importance  to  the  silk  industry. 


FIG.  93. — Oidium  lactis.  a,  b,  Dichotomous  branching  of  growing  hyphae;  c,  d.  g, 
simple  chains  of  oidia  breaking  through  substratum  at  dotted  line  x-y,  dotted  por- 
tions submerged;  e,  /,  chains  of  oidia  from  a  branching  outgrowth  of  a  submerged 
cell;  h,  branching  chain  of  oidia;  k,  /,  m,  n,  o,  p,  s,  types  of  germination  of  oidia  under 
varying  conditions;  /,  diagram  of  a  portion  of  a  colony  showing  habit  of  Oidium 
lactis  as  seen  in  culture  media.  (From  Bull.  82,  Bur.  Animal  Industry,  U.  S.  Dept. 
Agr.) 

Oidium  Lactis. — Oidium  lactis  is  very  widely  distributed  and 
is  almost  always  present  in  milk  and  milk  products,  and  in  brew- 
er's and  baker's  yeast,  and  it  is  an  especially  prominent  organism 
in  the  further  fermentation  of  acid  substances,  such  as  sauer- 


MOLDS    AND    YEASTS    AND    DISEASES    CAUSED   BY    THEM      237 

kraut,  sour  milk  and  cheese.  The  organism  is  especially  impor- 
tant in  the  ripening  of  Camembert  cheese.  It  grows  well  on  ordi- 
nary nutrient  gelatin.  The  colony  consists  of  a  loosely  woven, 
white  network  of  septate,  branched  and  anastomosing  threads, 


FIG.  94. — Oidium  albicans.     A  deep  colony  on  a  plate  culture  of  the  liquifying 
variety,  showing  chlamydospores.     (After  Plant.} 

chiefly  in  the  substratum  but  also  extending  into  the  air.  The 
peripheral  threads  are  divided  by  septa  to  form  chains  of  oval 
or  spherical  conidia. 

This  mold  may  be  readily  obtained  for  study  by  making  plate 
cultures  from  compressed  yeast. 


238  SPECIFIC  MICRO-ORGANISMS 

Oidium  Albicans  (Monilia  Candida). — The  thrush  fungus 
was  discovered  by  von  Langenbeck  in  1839  and  by  Berg  in  1841, 
but  the  popular  recognition  of  a  relation  between  this  disease  and 
a  mold  seems  to  have  preceded  this  discovery  by  many  years. 
Berg  (1841)  transferred  the  fungus  from  cases  of  thrush  to  healthy 
children  with  positive  results.  His  work  was  confirmed  by  numer- 
ous other  investigators  (1842-43).  Robin  (1847)  accurately 
described  the  parasite,  with  illustrations,  classed  it  as  an  oidium, 
and  gave  it  the  name  Oidiym  albicans  (1853).  Grawitz  (1877) 
obtained  the  first  pure  cultures  and  successfully  inoculated  rab- 
bits and  puppies  with  them. 

In  the  throat  lesion  as  well  as  in  cultures  the  organism  con- 
sists of  mycelial  threads  and  oval  yeast-like  cells.  It  grows  read- 


FIG.  95. — Oidium  albicans.     Mycelial  thread  with  four  ripe  chlamydospores;  and 
conidia  in  the  middle  of  the  picture.     (After  Plant.} 

ily  on  various  artificial  media  and  the  appearance  of  the  growth  is 
quite  variable,  not  only  because  of  the  proportional  relation 
between  the  oval  cells  and  the  threads,  but  also  in  pigmen- 
tation and  in  density  of  growth.  Two  varieties,  one  liquefying 
gelatin  and  producing  large  (5^)  oval  conidia,  and  the  other  failing 
to  liquefy  gelatin  and  producing  small  (2.5^)  spherical  conidia 
are  distinguished. 

Thrush  is  most  common  on  the  buccal  mucous  membrane  of 
young  infants,  but  it  also  occurs  on  the  vaginal  mucosa  of  preg- 
nant women,  and  it  may  attack  others  when  weakened  by  dis- 
ease, especially  diabetics.  The  disease  also  occurs  naturally  in 
birds,  calves  and  foals.  The  threads  of  the  mold  penetrate  the 
squamous  epithelium  and  even  enter  the  subepithelial  tissue, 
sometimes  penetrating  blood-vessels  and  giving  rise  to  metas- 


MOLDS   AND   YEASTS   AND   DISEASES   CAUSED  BY  THEM         239 

tases.  It  results  in  death  in  about  20  per  cent  of  the  cases  in 
infants.  The  predisposing  digestive  disorder  or  other  primary 
disease  is,  however,  usually  more  important  than  the  thrush,  and 
demands  first  consideration  in  treatment.  The  thrush  lesion  may 
be  carefully  removed  with  a  soft  swab  and  the  eroded  area  treated 
with  silver  nitrate,  o.i  per  cent.  Generalization  of  the  disease 
is  rare,  but  several  cases  have  been  observed.  Inoculation  of 
animals  (mice,  guinea-pigs,  puppies,  rabbits)  is  sometimes  success- 
ful, and  generalized  thrush  has  followed  intravenous  injection  of 
young  rabbits.  The  fungus  seems  to  exert  some  poisonous  action, 
in  addition  to  the  mechanical  effect  upon  the  tissues. 


FIG.  96. — Scutulum  of  favus  on  the  arm  of  a  man.     (After  Plant.) 

Achorion  Schoenleinii. — The  fungus  of  favus  was  discovered  by 
Schoenlein  in  the  skin  lesions  of  this  disease  in  1839,  two  years 
after  the  recognition  of  Botrytis  bassiana  as  the  cause  of  mus- 
cardine.  Remak  in  1845  grew  the  mold  on  slices  of  apple  and 
successfully  inoculated  his  skin  with  these  cultures.  He  named 
the  organism  Achorion  schoenleinii.  In  the  lesion  of  favus  the 
threads  of  the  fungus  are  found  growing  in  the  horny  layer  of  the 
epidermis,  usually  about  a  hair,  and  giving  rise  to  a  dry,  circular, 


240  SPECIFIC  MICRO-ORGANISMS 

yellow  crust  with  depressed  center,  the  "Scutulum.'"  By  macerat- 
ing this  crust  in  50  per  cent  antiformin  the  elements  of  the 
mold  are  made  clearly  visible  under  the  microscope.  In  the 
center  of  the  lesion  are  doubly  contoured  oval  or  rectangular 
conidia  3  to  8/*  by  3  to  4//,  single  and  in  chains.  The  mycelial 
threads  are  indistinguishable  in  the  center,  but  are  seen  at  the 
periphery  as  tubes  of  very  irregular  width,  refractive  with  granu- 
lar protoplasm,  often  branched  or  knobbed  at  the  end.  The 


FIG.  97. — Typical  scutulum  of  favus  in  a  mouse.     (After  Plant.) 

scutulum  in  its  interior  is  a  pure  culture  of  the  mold,  entirely  free 
from  other  organisms.  The  mold  also  grows  in  the  interior  of 
the  hair  shaft,  and  by  macerating  the  hair  in  alkali  the  fungus 
may  be  demonstrated  microscopically. 

Cultures  may  be  obtained  upon  various  media.  Plaut  recom- 
mends a  medium  containing  pepton  i  to  2  per  cent,  glycerin 
0.5  per  cent,  salt  0.5  per  cent  and  agar  2  per  cent,  without  meat 
extractives  or  any  addition  of  alkali.  The  cultures  are  incubated 
at  30°  C.  Mycelial  threads  and  numerous  conidia  are  produced. 

Inoculation  into  the  epidermis  of  mice  or  onto  the  human 


MOLDS    AND    YEASTS    AND    DISEASES    CAUSED   BY    THEM 


241 


skin  gives  rise  to  typical  lesions.  Intravenous  injection  into 
rabbits  is  usually  followed  by  a  pseudo-tuberculosis  in  the  lungs, 
sometimes  fatal.  Similar  skin  lesions  occur  naturally  in  various 
animals,  and  the  molds  there  present  are  very  similar  to,  if  not 


FIG.  98. — Achorion  schoenleinii.    Colony  developing  from  a  favus  scale.    End,  endo- 
conidia  on  submerged  hyphae.     Ect,  ectospores  on  aerial  hyphae.     (After  Plant.} 

specifically    identical    with,    Achorion    schoenleinii.     The    exact 
relationships  of  the  parasites  are  not  very  exactly  settled  as  yet. 

Microsporon  Audouini. — This  mold  is  found  growing  in  the 
hair-shaft  in  alopecia  areata.     If  the  hair  be  pulled  out  it  breaks 

16 


242 


SPECIFIC  MICRO-ORGANISMS 


near  the  lower  end  and  the  oval  conidia  and  jointed  threads  of 
the  parasite  may  be  demonstrated  by  macerating  this  broken 
end.  The  disease  is  very  contagious,  chronic  and  resistant  to 

treatment,  but  proceeds  without  inflam- 
mation or  subjective  symptoms,  the 
conspicuous  sign  being  loss  of  the  hair. 
Cultures  grow  slowly  and  are  snow 
white.  Animal  inoculation  is  rarely 
successful. 

Microsporon  Furfur. — This  mold  is 
found  in  the  superficial  layer  of  the  skin 
in  pityriasis  versicolor,  as  short  thick 
hyphae  3  to  ^  wide  by  7  to  13,"  long, 
together  with  abundant  doubly  con- 
toured single  conidia.  Pityriasis  versi- 
color occurs  most  frequently  on  the 
skin  of  the  chest  and  is  one  of  the  com- 
monest affections  of  the  skin. 

Tricophyton  Acuminatum. — The 
mold  invades  the  hair  shaft  and  causes 
it  to  break  off  close  to  the  surface  of 
the  skin.  In  such  a  hair  long  chains 
of  oval  cells  of  the  parasite  may  be  seen. 
The  parasite  also  attacks  the  skin  and 
produces  ringworm.  Several  other  spe- 
FIG.  gg.—Sporotrichum  cies  of  tricophyton  are  distinguished. 

schencki.    Cultures  on  the    Tnese  parasites   are  concerned   in  the 
glucose-pepton  agar  of  Sabour-  f 

aud.    (After  Gougerot.)  causation  of  barber's  itch,  eczema  mar- 

ginatum,  tinea  cruris,  and  other  skin 
affections  of  this  type. 

Sporotrichum  Schencki. — Schenck,  at  Baltimore  in  1898,  de- 
scribed this  parasitic  mold  which  he  found  in  the  lesions  of  a 
peculiar  disease,  beginning  as  a  localized  ulcer,  with  later  involve- 
ment of  the  neighboring  lymph  glands,  in  which  cold  abscesses 
formed  and  opened  to  the  exterior.  A  second  similar  case  was 


AND  YEASTS  AND  DISEASES  CAUSED  BY  THEM 


243 


described  by  Hektoen  and  Perkins.     Ruediger1  has  reported  a 
large  series  of  cases  of  sporotrichosis  and  the  references  to  American 


FIG.  ioo. — Sporotrichum  schenki.  _  Various  forms  of  mycelum  with  and  without 
conidia.     (After  Gougerot.} 

literature  will  be  found  in  his  paper.     The  organisms  are  not 

1  Journ.  Infect.  Diseases,  1912,  Vol.  XI,  pp.  193-206. 


244  SPECIFIC   MICRO-ORGANISMS 

readily  found  in  the  pus  by  microscopic  examination  and  seem 
to  exist  there  only  as  conidia.  In  cultures  a  branching  mycelium 
with  clusters  of  conidia  is  produced.  Dogs  are  susceptible  to 
inoculation. 

Sporotrichium  Beurmanii. — De  Beurmann  and  Ramond  at 
Paris  in  1903  found  this  parasite  in  a  case  of  lymphangitis.  It 
seems  to  be  different  from  the  organism  described  by  Schenck 
but  may  ultimately  prove  to  be  the  same  species. 

Saccharomyces  Cerevisiae. — This  organism  is  ,the  type  of 
the  true  yeasts.  The  cell  is  spherical  or  ovoid,  and  multiplies 
by  budding.  Endospores  are  produced,  usually  four  to  eight 
in  a  single  cell,  indicating  a  rather  close  relationship  to  the  molds. 
The  organism  is  found  widely  distributed,  especially  on  fruits 
and  sugar-containing  substances.  It  has  been  used  for  centuries  in 
the  leavening  of  bread  and  in  the  alcoholic  fermentations.  Varie- 
ties of  the  species  are  distinguished  by  differences  in  physiological 
characters,  and  especially  in  respect  to  the  amounts  of  alcohol 
which  they  produce. 

Material  for  study  may  be  obtained  from  commercial  com- 
pressed yeast,  which  contains  vegetating  cells  of  saccharomyces 
along  with  other  organisms,  or  from  commercial  dried  yeast  in 
which  the  spores  are  present.  Pure  cultures  may  be  obtained  by 
plating  on  gelatin.  True  yeasts  also  occur  in  the  gastric  juice  at 
times  and  seem  to  be  able  to  multiply  in  the  stomach  when  the 
acidity  of  the  gastric  juice  is  diminished. 

Blastomyces  Dermatidis. — Doubly  contoured  yeast-like  cells 
in  human  tissues  were  first  discovered  by  Busse  and  Buschke1  in 
1 894,  in  a  case  presenting  abscesses  in  the  bones  and  internal  organs 
together  with  lesions  of  the  skin.  They  obtained  cultures  of  the 
organism  and  classed  it  as  a  yeast.  About  the  same  time  Gil- 
christ2  independently  observed  similar  organisms  in  cases  of 
dermatitis  at  Baltimore.  The  organisms  have  been  most  thor- 
oughly studied  by  Ricketts.3  Most  of  the  cases  have  been  ob- 

1  Deutsch.  med.  Wochenschr.,  1895,  Nr.  3. 

2  Gilchrist:  Johns  Hopkins  Hosp.  Kept.,  Vol.  I,  p.  209,  1896. 
3Journ.  Med.  Research,  Vol.  VI,  No.  3. 


MOLDS    AND    YEASTS    AND    DISEASES    CAUSED   BY    THEM 


245 


served  in  the  United  States,  at  Baltimore,  at  Chicago  and  in  Cali- 
fornia. One  type  of  the  parasite  appears  to  multiply  in  the  tissues 
by  a  process  of  budding  (Blastomycetic  dermatitis,  Blastomycosis) 
while  in  other  cases,  particularly  those  from  California,  the 
spherical  bodies  found  in  the  tissue  seem  to  multiply  by  endog- 
enous spore  formation,  an  appearance  which  at  first  suggested 
the  protozoal  nature  of  the  parasite  and  lead  to  the  use  of  the 


FIG.  101. — Doubly  contoured  organisms  found  in  oidiomycosis  (blasto  mycosis). 
(From  Buschke  after  Hyde  and  Montgomery.) 

unfortunate  term,  Coccidioidal  granuloma.  On  glucose  agar,  the 
parasites  usually  grow  without  difficulty  and  the  growth  resem- 
bles that  of  an  oidium,  often  with  abundant  aerial  hyphae.  Inoc- 
ulation of  guinea-pigs  with  pus  or  with  cultures  is  usually  fol- 
lowed by  formation  of  abscesses  in  which  the  typical  spherical  or 
ovdid  parasites  may  be  found.  The  tissue  changes  have  been 
mistaken  for  tuberculosis.  Further  investigations  are  required 
to  determine  the  specific  relationships  of  the  parasites  found  in 
different  cases. 


CHAPTER  XV. 
TRICHOMYCETES. 

The  trichomycetes  or  higher  bacteria  are  intermediate  in 
morphological  characters  between  the  molds  and  the  lower  bac- 
teria. They  resemble  the  molds  in  the  formation  of  long  threads, 
sometimes  branching  and  interlacing  to  produce  a  network,  and 
in  the  formation  of  oval  or  spherical  conidia  constricted  off  from 
the  ends  of  the  threads.  They  resemble  the  lower  bacteria  in 
their  small  transverse  diameter,  the  delicacy  of  their  structure 
and  their  mode  of  life.  Petruschy1  recognizes  four  genera, 
Actinomyces,  Streptothrix,  Cladothrix  andLeptothrix. 

Actinomyces  Bovis. — Bollinger  in  1877  studied  the  lumpy- 
jaw  disease  of  cattle  and  described  this  parasite  which  occurs 
in  the  lesions.  Israel,  in  the  following  year,  found  the  organism 
in  granulomatous  lesions  in  man.  The  infection  also  occurs  in 
horses,  sheep,  swine  and  dogs.  In  the  tissues  and  in  the  purulent 
discharge  from  the  lesions,  the  organism  occurs  in  small  yellowish 
masses,  sometimes  visible  to  the  naked  eye  but  usually  smaller 
(10  to  2oo/*  in  diameter).  Such  a  mass  is  a  single  colony  of  the 
parasite  or  a  conglomerate  of  several  colonies.  The  colony  is  a 
dense  network  of  threads  in  the  center,  with  radially  arranged 
threads  about  the  periphery,  most  of  the  latter  being  swollen, 
club-shaped,  at  their  free  ends.  Spherical  bodies  may  also  be 
present,  but  whether  these  are  conidia  or  degeneration  forms  of 
the  parasite  is  uncertain.  The  organism  is  Gram-positive. 

Inoculation  of  pus  or  bits  of  tissue  containing  the  parasite 
from  one  animal  into  another  usually  fails  to  transmit  the  dis- 
ease, although  positive  results  have  been  obtained  in  a  few  in- 
stances. Attempts  at  culture  have  failed  also  in  many  instances, 

1  Kolle  and  Wassermann:  Handbuch,  1912,  Vol.  V,  p.  270. 

46 


TRICHOMYCETES  247 

and  the  difficulty  here  seems  to  depend  in  part  upon  the  oxygen 
requirements  of  the  organism.  The  material  for  culture  should 
be  obtained  from  uncontaminated  tissue  containing  the  fungus. 
If  this  is  impossible,  the  granule  of  actinomyces  should  be  washed 
in  several  changes  of  sterile  salt  solution,  then  crushed  between 
sterile  glass  slides  or,  better,  ground  up  in  a  sterile  mortar  with 
a  small  amount  of  sterile  sand.  A  series  of  dilution  cultures 
should  then  be  made  in  tall  tubes  of  melted  glucose  agar  cooled  to 
45°  C.,  the  tubes  chilled  in  cold  water  and  incubated  at  37°  C. 


FIG.  102. — Actinomyces  bovis.     The  ray-fungus  from  cow.     (Diagrammatic.) 


Colonies  of  the  fungus  may  be  expected  to  develop  some  distance 
below  the  surface  of  the  agar.  Wolf  and  Israel  were  able  to 
reproduce  the  disease  in  animals  (rabbits  and  guinea-pigs)  by  the 
inoculation  of  pure  cultures.  More  recently  Harbitz  and  Gron- 
dahl1  isolated  twenty-seven  strains  of  actinomyces,  but  their 
inoculation  experiments  were  wholly  negative.  It  would  appear 
that  other  factors  are  essential  to  the  development  of  actinomy- 
ces in  addition  to  the  inoculation  of  the  specific  parasite.  Many 
authors  regard  the  presence  of  bits  of  straw  or  sharp  grains  in 
wounds  of  the  mucous  membrane  of  the  mouth  or  pharynx  as 
important  elements  in  predisposing  to  infection  with  actinomyces. 

1  Amer.  Journ.  Med.  Sciences,  1911,  Vol.  CXLII,  pp.  386-395. 


248  SPECIFIC   MICRO-ORGANISMS 

The  disease  shows  little  or  no  tendency  to  be  transmitted  from 
animal  to  animal  in  a  herd.  Several  varieties  of  actinomyces 
have  been  described,  and  possibly  more  than  one  species  will 
eventually  be  recognized. 

Streptothrix  Madurae. — Kanthack  (1892)  and  Gemy  and 
Vincent  (1892)  discovered  the  fine  mycelial  threads  in  pus  from 
cases  of  Madura  foot.  Granules  about  the  size  of  a  pin-head  occur 
in  the  pus,  and  under  the  microscope  these  are  found  to  consist  of 
a  network  of  threads  i  to  1.5^  in  thickness,  arranged  radially 
at  the  periphery  and  presenting  somewhat  swollen  ends.  These 
granules  are  white  in  some  cases,  yellow,  red  and  black  in  others. 
The  nature  of  the  disease  seems  to  be  the  same  in  all  cases,  but 
the  micro-organisms  are  apparently  not  the  same,  that  found  in 
the  black  variety  probably  representing  a  distinct  species. 
Cultures  may  be  obtained  by  inoculating  the  pus,  collected 
without  contamination,  into  several  flasks  of  sterilized  hay  in- 
fusion, and  shaking  daily  to  insure  abundant  oxygen  supply. 
It  also  grows  upon  other  media.  Gelatin  is  not  liquefied.  The 
growth  is  made  up  of  interwoven,  slender  branching  threads 
about  i/*  in  thickness.  Spores  (conidia)  capable  of  resisting  a 
temperature  of  75°  C.  for  five  minutes  are  produced  at  the  sur- 
face of  the  culture.  Inoculation  of  animals  usually  gives  nega- 
tive results,  but  Musgrave  and  Klegg1  have  succeeded  in  infecting 
monkeys. 

The  disease,  Mycetoma  or  Madura  foot,  is  a  localized  chronic 
inflammation,  almost  painless,  and  usually  involving  the  foot, 
the  hand  or  some  exposed  portion  of  the  body.  The  disease 
involves  the  tissues  by  direct  extension,  attacking  the  bones  as 
well  as  the  soft  tissues.  It  usually  remains  localized  to  one 
extremity. 

The  black  variety  of  Madura  foot  is  due  to  a  different  organ- 
ism, the  threads  of  which  are  3  to  8/*  in  thickness.2  This  organ- 

1  Philippine  Journ.  of  Science,  1907,  Vol.  II,  pp.  477-512;   A  complete  bibli- 
ography by  Polk  is  included. 

2  Wright:  Journ.  of  Exp.  Medicine,  Vol.  Ill,  pp.  421-433. 


TRICHOMYCETES  249 

ism  seems  to  be  an  aspergillus,  and  has  been  named  Madurella 
mycetori. 

Streptothrices  have  also  been  found  in  abscesses  of  the  brain  and 
in  chronic  disease  of  the  lung  clinically  resembling  tuberculosis 
in  man.  Many  of  them  are  Gram-positive  and  some  are  rela- 
tively acid-proof  when  stained  with  carbol-fuchsin.  Such  acid- 
proof  forms  are  common  in  the  feces  of  cattle  where  short  seg- 
ments of  them  may  be  mistaken  for  tubercle  bacilli.  Organisms 
of  this  type  are  very  abundant  in  the  soil,  which  is  doubtless 
their  natural  habitat. 

Cladothrix. — The  cladothrix  forms  resemble  the  strepto- 
thrices  very  closely  but  the  cells  of  the  threads  do  not  branch. 
The  apparent  branching  of  the  threads  is  explained  as  due  to  a 
transverse  division  of  the  thread  with  continuing  growth  of  the 
one  free  end  which  pushes  out  beyond  the  other,  giving  rise  to 
the  appearance  of  branching  or  so-called  " false  branching." 
Organisms  of  this  type  have  been  described  as  occurring  in  ab- 
scesses of  the  brain  and  in  other  parts  of  the  body.  The  dis- 
tinction from  streptothrix  has  not  always  been  clearly  made. 

Leptothrix  Buccalis. — This  is  a  normal  inhabitant  of  the 
mouth  cavity.  It  consists  of  slender  filaments  which  do  not 
branch.  The  organism  has  been  found  in  abundance  in  small 
white  patches  on  the  tonsils,  where  it  sometimes  causes  a  very 
chronic  but  mild  inflammation.  Artificial  culture  of  the  organ- 
ism ordinarily  results  in  failure.  Arustamoff1  appears  to  have 
obtained  it  on  a  neutral  or  acid  agar  inoculated  with  leptothrix 
from  urine. 

1  Kolle  and  Wassermann:  Handbuch,  1912,  Bd.  V,  S.  290. 


CHAPTER  XVI. 

THE  COCCACE^:  AND  THEIR  PARASITIC  RELATION- 
SHIPS. 

Diplococcus  Gonorrheae. — The  gonococcus  was  discovered 
by  Neisser1  in  1879  in  the  discharge  of  acute  urethritis  and  he 
recognized  its  probable  causal  relationship  to  the  disease.  Cul- 
tures were  first  obtained  by  Bumm2  in  1885  and  he  proved  the 
relationship  by  inoculating  the  human  urethra  with  his  cultures. 
The  organism  naturally  lives  and  multiplies  only  in  the  human 
body  and  is  the  microbic  cause  of  gonorrhea  and  many  of  its 
complicating  inflammations. 

The  gonococcus  is  found  in  both  the  serum  and  the  poly- 
nuclear  cells  of  the  purulent  discharge,  usually  in  pairs  with  the 
adjacent  surfaces  flattened.  The  long  diameter  of  the  pair  is 
about  i. 25/4.  It  stains  readily,  best  perhaps  with  Loffler's 
methylene-blue.  It  is  decolorized  when  stained  by  Gram's 
method,  a  fact  of  great  importance  in  the  quick  recognition  of 
the  organism.  The  staining  procedure  has  to  be  carefully  carried 
out  and  a  beginner  should  practice  upon  cultures  of  the  gonococcus 
and  upon  samples  of  gonorrheal  pus  and  staphylococcus  pus 
before  placing  too  much  reliance  upon  the  appearance  of  his 
Gram-stained  preparation.  The  reaction  to  the  Gram  stain, 
together  with  the  remarkably  characteristic  appearance  of  the 
pus  cell  full  of  diplococci  are  usually  sufficient  for  the  recogni- 
tion of  the  organism  in  acute  urethritis. 

Cultures  of  the  gonococcus  were  obtained  by  Bumm  on  coagu- 
lated human  blood  serum.  Wertheim3  employed  serum  agar 

1  Neisser:  Centralbl.  f.  d.  med.  Wissenschaft,  1879,  Bd.  XVII,  S.  497-500. 

2  Bumm:  Deutsche  med.  Wochenschr.,  1885,  Bd.  II,  S.  910  and  911. 

3  Deutsche  med.  Wochenschr.,  1891,  Bd.  XVII,  S.  958;  S.  1351  and  1352. 

250 


COCCACE^E    AND    THEIR    PARASITIC    RELATIONSHIPS 


251 


made  by  mixing  human  blood  serum  at  40°  C.,  one  part,  with 
ordinary  nutrient  agar  melted  and  cooled  to  40°  C.,  two  parts. 
The  medium  may  be  inclined  in  tubes  or  may  be  employed  for 
plating.  Human  ascitic  fluid  or  hydrocele  fluid  is  just  as  good 
as  blood  serum.  A  large  drop  of  pus  from  an  acute  urethritis 
should  be  mixed  with  2  to  3  c.c.  of  serum  or  ascitic  fluid  in  a 
test-tube  and  from  this,  dilutions  made  to  a  second  and  a  third 
tube.  The  contents  of  a  tube  of  agar  (5  to  6  c.c.),  previously 
melted  and  cooled  to  about  40°  C.,  is  then  added  to  each  tube  of 


FIG.  103. — Gonococci  and  pus-cells.     Xiooo. 

serum,  mixed  thoroughly  and  poured  into  Petri  dishes  to  solidify. 
At  37°  C.,  colonies  appear  within  24  hours  and  at  the  end  of  this 
time  measure  about  i  mm.  in  diameter.  The  colony  is  circular, 
grayish-blue  and  transparent  and  of  a  mucoid  consistency. 
The  individual  cocci  disintegrate  rapidly,  even  within  the  first 
24  hours  at  the  center  of  the  colony,  and  for  microscopic  study, 
simple  staining  and  staining  by  Gram's  method,  cultures  5  to  10 
hours  old  are  recommended.  Even  under  favorable  conditions 
the  gonococcus  ordinarily  dies  out  in  the  culture  tube  in  about  a 
week,  although  exceptionally  it  may  survive  for  three  weeks. 


252  SPECIFIC   MICRO-ORGANISMS 

It  should  be  transplanted  every  few  days  and  a  large  quantity 
of  growth  must  be  transferred.  When  transplanted  from  vigor- 
ous cultures  to  plain  agar  the  gonococcus  grows  for  a  few  days, 
but  it  cannot  be  successfully  propagated  for  any  length  of  time 
on  ordinary  media. 

The  gonococcus  is  very  sensitive  to  drying  and  to  tempera- 
tures above  40°  C.  It  is  usually  impossible  to  recover  it  from 
dried  pus,  but  in  moist  material  it  may  live  for  i  to  24  hours. 
The  organism  is  easily  killed  by  chemical  germicides,  of  which 
silver  nitrate  is  probably  the  most  effective. 

Inoculation  of  animals  in  the  urethra  or  on  the  conjuctiva 
is  without  result.  Intraperitoneal  injection  of  cultures  into 
white  mice  or  guinea-pigs  usually  kills  the  animals  in  24  hours 
and  the  gonococci  can  be  recovered  from  the  peritoneal  fluid 
and  the  heart's  blood.  These  effects  seem  to  be  due  to  toxins  of 
the  injected  material  rather  than  actual  infection.  The  specific 
poisons  seem  to  be  intracelluar  and  set  free  upon  disintegration 
of  the  organism.  The  poison  withstands  heating  to  100°  C.  for 
hours.  Inoculation  of  the  human  urethra  with  cultures  of  the 
gonococcus  has  been  repeatedly  done  and  has  resulted  nearly  al- 
ways in  the  production  of  typical  gonorrhea. 

Gonnorrhea  has  been  recognized  as  a  contagious  disease 
since  the  dawn  of  history.  The  most  important  forms  are  (i) 
urethritis  with  tendency  to  extension  in  the  female  to  the  cervix 
uteri,  oviducts  and  peritoneum,  and  iri  the  male  to  the  prostate, 
seminal  vesicles,  and  epididymis,  and  in  both  sexes  to  the  blood 
stream,  heart  valves  and  joints;  (2)  conjunctivitis  and  keratitis 
leading  to  scarring  of  the  cornea  and  permanent  blindness; 
(3)  valvo-vaginitis  in  girl  babies,  an  exceedingly  contagious 
disease,  especially  in  hospital  wards.  The  disease  tends  to 
become  chronic  and  eventually  latent,  that  is,  the  symptoms 
subside  but  the  micro-organisms  remain  alive  in  certain  loca- 
tions, such  as  the  prostate  in  the  male  and  the  cervix  uteri  in 
the  female.  The  acute  inflammation  may  be. followed  by  scars' 
resulting  in  strictures  of  the  urethra  or  occlusion  of  the  epididy- 


COCCACE^:    AND     THEIR    PARASITIC    RELATIONSHIPS          253 

mis.  In  the  female,  pyosalpinx  is  a  not  unusual  complication. 
Secondary  infection  with  staphylococci  is  common  in  chronic 
gonorrhea. 

Specific  diagnosis  by  finding  gonococci  usually  presents  no 
difficulties  in  acute  inflammations  of  the  genital  tract,  in  which 
the  characteristic  groups  of  Gram-negative  intracellular  diplococci 
are  practically  diagnostic.  In  chronic  cases  and  in  extra-genital 
inflammations  the  diagnosis  presents  greater  difficulty.  Both 
microscopic  and  cultural  examinations  should  be  made  and  if 
negative  they  should  be  repeated  many  times.  Even  repeated 
failure  to  find  the  gonococcus  by  these  methods  does  not  justify 
the  positive  assertion  that  it  is  absent.  Specific  diagnosis  by 
the  method  of  complement  fixation  has  been  developed  by 
Schwartz  and  McNeill. 1  The  antigen  is  prepared  from  several  cul- 
ture strains  of  the  gonococcus  and  in  all  other  respects  the  test  is 
similar  to  the  Wassermann  test  for  syphilis.  Irons2  has  employed 
a  cutaneous  test,  using  a  glycerin  extract  of  gonoc<  -cci.  The  tech- 
nic  is  similar  to  that  of  the  von  Pirquet  test  for  tuberculosis. 

The  prevalence  of  gonorrhea  throughout  the  civilized  world 
is  much  greater  than  has  been  popularly  supposed.  Erb,  in  a 
study  of  2000  males  among  private  patients  of  the  middle  and 
better  classes,  found  a  history  of  gonorrhea  in  50  per  cent.  Many 
other  students  of  the  disease  disagree  with  Erb,  regarding  his 
figures  as  much  too  low.  Among  women  in  German  obstetrical 
hospitals,  largely  from  the  poorer  class,  gonorrhea  is  present  in 
10  to  30  per  cent.  The  danger  to  the  eyes  of  the  new-born 
infant  is  now  overcome  by  the  use  of  silver  nitrate  in  the  eyes 
when  they  are  first  cleansed.  The  general  prevention  and  re- 
striction of  gonorrheal  infection  is  engaging  more  and  more  the 
serious  attention  of  thoughtful  citizens,  and  it  is  already  recog- 
nized as  a  sanitary  problem  of  the  first  magnitude. 

Diplococcus  Meningitidis. — Weichselbaum  in  1887  examined 
the  cerebrospinal  fluid  in  six  sporadic  cases  of  meningitis  and 

1  Amer.  Joitrn.  med.  Sciences,  1912,  Vol.  CXLIV,  pp.  815-826. 

2  Jour n.  Infec.  Diseases,  1912,  Vol.  XI,  pp.  77-93. 


254  SPECIFIC  MICRO-ORGANISMS 

found  in  all  of  them  a  very  definite  Gram-negative  intracellular 
diplococcus,  the  meningococcus.  He  obtained  cultures  but  his 
animal  inoculatons  all  gave  negative  results.  Jaeger  in  1895 
seems  to  have  found  a  similar  organism  in  fourteen  cases  of 
epidemic  meningitis  and  Huebner  in  1896  apparently  found  it 
in  five  cases.  The  cultural  work  of  these  authors  seems  to  be 
unreliable  as  their  cultures  were  Gram-positive.  More  conclu- 
sive confirmation  of  the  relation  of  this  organism  to  epidemic 
meningitis  was  furnished  by  Councilman,  Mallory  and  Wright1 
in  1898. 

The  meningococcus  is  found  in  the  bodies  of  patients  suffering 
from  meningitis,  occasionally  on  the  nasal  mucous  membrane 
of  healthy  persons  and  of  cases  of  rhinitis,  and  very  rarely  in 
other  situations.  In  cerebrospinal  meningitis  the  organism  is 
present  in  the  cerebrospinal  fluid,  in  the  meninges,  often  on  the 
nasal  and  pharyngeal  mucous  membrane,  sometimes  in  the 
blood  and  on  the  conjunctivae,  and  rarely  in  the  urethra,  where 
it  may  be  mistaken  for  the  gonococcus.  It  is  usually  found 
without  difficulty  in  the  cerebrospinal  fluid  in  the  first  few  days 
of  the  disease,  but  may  be  very  difficult  to  find  at  a  later  stage. 

The  organism  is  found  for  the  most  part  inside  polynuclear 
leukocytes  and  in  its  form,  size,  arrangement  and  behavior  to 
the  Gram-stain  resembles  very  closely  the  gonococcus.  The 
outline  of  the  cocci  is  often  somewhat  hazy,  suggesting  possible 
disintegration,  and  this  sometimes  makes  their  recognition 
somewhat  difficult  in  microscopic  preparations  of  cerebrospinal 
fluid.  Cultures  are  best  made  on  ascitic-fluid  agar  or  blood 
agar,  upon  which  small  dew-drop  colonies  appear  in  24  hours 
at  37°  C.  The  color  of  blood  is  unaltered  by  the  growth.  Cul- 
tures may  be  obtained  on  Loffler's  blood  serum,  although  ascitic- 
fluid  agar  is  probably  the  best  medium  for  continued  culti- 
vation. The  meningococcus  grows  more  luxuriantly  than  the 
gonococcus,  as  a  rule,  and  adapts  itself  more  readily  to  growth 

1  Report  of  the  Mass.  Bd.  of  Health  on  Epidemic  Cerebrospinal  Meningitis, 
etc.,  Boston,  1898. 


COCCACE.E    AND     THEIR    PARASITIC     RELATIONSHIPS  255 

on  ordinary  media,  but  its  cells  disintegrate  rapidly  in  the  colony, 
which  is  viscid.  In  nearly  every  respect  it  resembles  very  closely 
the  gonococcus. 

Intraperitoneal  inoculation  of  white  mice  and  of  guinea-pigs 
usually  results  in  fatal  peritonitis  and  the  organism  can  be  recov- 
ered from  the  heart's  blood.  Intraspinal  inoculation  of  monkeys 
with  large  doses  causes  typical  meningitis  with  symptoms  similar 
to  those  of  the  disease  in  man.  In  man  the  disease  is  undoubtedly 
transmitted  very  largely  by  coccus-carriers,  healthy  people  or 
people  with  slight  pharyngitis  or  rhinitis,  who  carry  the  virus 
on  their  mucous  membranes  and  distribute  it. 

Antimeningococcus  serum  is  prepared  by  immunizing  horses 
with  a  mixture  of  many  typical  and  atypical  meningococcus 
cultures  injected  subcutaneously.  At  first  the  cultures  are 
killed  by  heat  before  injection,  and  only  one  or  two  loopfuls  are 
given.  The  dose  is  increased  and  repeated  every  8  to  10  days 
until  the  growth  on  two  Petri  dishes  is  being  injected.  Living 
cultures  are  then  given,  and  finally  old  cultures  which  have 
disintegrated  are  also  used.  The  serum  is  used  after  the  horse 
has  been  treated  for  8  to  10  months.  Jochmann  showed  that 
the  subcutaneous  injection  of  the  serum  is  without  effect  upon 
meningitis  in  monkeys  but  that  when  introduced  into  the  spinal 
canal  is  specifically  curative.  Flexner1  and  his  co-workers 
have  studied  this  very  fully  and  there  can  no  longer  be  any  ques- 
tion of  the  value  of  the  serum  in  the  treatment  of  meningococcus 
meningitis. 

Cerebrospinal  fluid  is  obtained  by  Quincke's  puncture.  For 
children  a  needle  4  cm.  long  and  with  a  lumen  of  i  mm.  is  intro- 
duced near  the  median  line  upward  and  forward  so  as  to  enter 
the  spinal  canal  between  the  second  and  third  or  the  third  and 
fourth  lumbar  vertebrae.  From  20  to  50  c.c.  of  fluid  may  be  with- 
drawn if  it  comes  away  under  pressure,  and  then  the  curative  serum 
is  injected  through  the  same  needle.  The  fluid  withdrawn  should 
be  examined  to  establish  the  presence  of  meningitis  and  its 

Flexner:  Harbin  lectures.     Journ.  State  Medicine,  1912,  Vol.  XX,  pp.  257-270. 


256 


SPECIFIC   MICRO-ORGANISMS 


variety.  In  general  the  examination  includes  a  macroscopic 
examination  and  description  of  the  appearance  of  the  sample,  a 
microscopical  numerical  count  of  the  cells  present,  chemical 
examination  of  the  cell-free  fluid  for  excessive  protein1  content, 
microscopic  and  cultural  examination  of  the  sediment  for  bacteria 
and  of  the  filmy  clot  which  may  form  after  standing  an  hour  or 


f 


* 


:« 


FIG.  104. — Meningococcus  in  spinal  fluid.     (After  Hiss  and  Zitisser.) 


so  for  tubercle  bacilli,  and  sometimes  it  includes  the  Wassermann 
reaction.  In  meningococcus  meningitis  the  cell  count  is  generally 
above  100  per  cu.  mm.,  and  most  of  the  cells  are  polynuclear 
leukocytes.  Within  these  cells  the  meningococci  may  or  may  not 
be  found.  In  case  of  doubt,  plate  cultures  on  blood-agar  and 

1  Noguchi's  test:  To  0.5  c.c.  of  blood-free  fluid  add  i  c.c.  iq  per  cent  butyric 
acid,  boil;  add  0.2  c.c.  normal  NaOH  and  boil  again.  Set  aside  to  cool.  A  floc- 
culent  precipitate  indicates  an  increase  in  the  globulin  content. 


COCCCAE.E    AND    THEIR    PARASITIC    RELATIONSHIPS          257 

ascitic-fluid  agar  should  be  made.  The  recognition  of  a  Gram- 
negative  intracellular  diplococcus  in  the  fluid  is  sufficient  for  a 
tentative  diagnosis,  and  the  appearance  of  characteristic  colonies 
on  the  plates  may  be  considered  conclusive. 

Diplococcus  (Micrococcus)  Catarrhalis. — This  organism  is 
commonly  present  on  the  mucous  membrane  of  the  upper  air 
passages,  especially  in  catarrhal  inflammations.  It  is  usually 
seen  as  a  Gram-negative  intracellular  diplococcus  not  to  be 
distinguished  microscopically  from  the  meningococcus  or  gono- 
coccus.  In  examining  material  from  the  air  passages  this  organ- 
ism has  to  be  considered.  It  is  readily  distinguished  by  cultural 
methods.  On  ascitic-fluid  agar  the  colony  is  dry  and  brittle, 
quite  different  from  the  meningococcus  or  gonococcus.  Further- 
more, it  grows  readily  at  once  on  ordinary  agar. 

Diplococcus  Pneumoniae. — Sternberg  in  1880  injected  the 
saliva  of  healthy  persons  into  rabbits  and  produced  a  rapidly 
fatal  bacteremia  with  abundant  lance-shaped  diplococci  in  the 
blood  and  internal  organs  of  the  animal.  Pasteur,  independently 
and  at  about  the  same  time,  injected  the  saliva  of  a  boy  suffering 
from  rabies  into  rabbits  with  a  similar  result.  The  organism 
was  spoken  of  as  the  diplococcus  of  sputum  septicemia  or  the 
septicemic  microbe  of  saliva.  Koch  in  1881  demonstrated  the 
organism  microscopically  in  sections  of  lung.  Friedlaender 
(1882-1884)  found  the  organism  microscopically  in  a  large 
number  of  cases  of  pneumonia  and  accurately  described  its  form, 
the  capsules  and  staining  properties.  His  cultures,  however, 
which  were  made  on  gelatin  at  room  temperature,  brought  to 
development  not  the  pneumococcus  but  a  wholly  different  organ- 
ism which  he  believed  to  be  identical  with  it,  Friedlaender's 
pneumobacillus.  A.  Fraenkel  obtained  the  first  undoubted  pure 
cultures  on  solidified  blood  serum,  proved  the  identity  of  the  organ- 
ism in  pneumonia  with  that  of  normal  saliva  seen  by  Sternberg 
and  Pasteur,  and  distinguished  it  absolutely  from  the  pneumo- 
bacillus of  Friedlaender.  He  also  succeeded  in  producing  typical 
pneumonia  by  injecting  cultures  of  moderate  virulence  intrave- 
17 


258  SPECIFIC  MICRO-ORGANISMS 

nously  into  rabbits.  Recently  Lamar  and  Meltzer1  have  induced 
typical  lobar  pneumonia  in  dogs  by  introducing  cultures  of  the 
pneumococcus  into  the  bronchi. 

The  pneumococcus  is  somewhat  variable  in  form.  In  the 
animal  body  it  occurs  in  pairs  of  lance-shaped  individuals  with 
the  points  directed  away  from  each  other,  and  the  pair  is  surrounded 
by  a  thick  gelatinous  capsule.2  The  organism  is  always  Gram- 
positive.  In  cultures  the  capsules  are  less  well  developed  and 
often  cannot  be  demonstrated  at  all.  The  individuals  are  often 


FIG.  105. — Pneumococcus,  showing  capsule,  from  pleuritic  fluid  of  infected  rabbit, 
stained  by  second  method  of  Hiss. 

less  pointed  and  frequently  resemble  short  bacilli  in  form.     They 
may  remain  attached  together  in  chains  of  six  to  eight  cells. 

Cultures  may  be  obtained  on  ordinary  media  but  they  are 
prone  to  die  out  quickly.  Blood-agar,  serum  agar  or  ascitic-fluid 
agar  are  the  best  solid  media,  but  even  with  these  weekly  trans- 
plantation is  usually  necessary.  Broth  to  which  serum  or  ascitic 

1  Journ.  Exp.  Med.,  1912,  Vol.  XV,  pp.  133-148. 

2  In  demonstrating  the  capsules,  the  method  of  Hiss  gives  excellent  results. 
Spread  some  blood  or  tissue  juice  on  a  cover -glass  and  as  soon  as  the  film  of  moisture 
has  disappeared,  fix  the  preparation  by  heat.     Then  stain  with  hot  aqueous  gentian 
violet  and  wash  off  the  dye  with  a  20  per  cent  solution  of  copper  sulphate.     Examine 
in  the  copper  solution.     Blot  the  preparation,  dry  it  in  air  and  mount  in  balsam. 


COCCACE.E    AND    THEIR    PARASITIC    RELATIONSHIPS  259 

fluid  has  been  added  forms  an  excellent  medium.  There  is  prac- 
tically no  growth  below  25°  C.  On  blood  agar,  the  colony  js 
surrounded  by  a  zone  of  greenish  discoloration,  a  character  of 
great  value  in  the  early  recognition  of  the  pneumococcus  isolated 
from  the  body.  The  virulence  of  the  microbe  diminishes  very 
rapidly  in  artificial  culture.  Virulent  material  is  best  kept  in 
stock  by  preserving  in  a  desiccator  dried  blood  taken  from  a 
rabbit  dead  of  pneumococcus  infection.  The  fluid  blood  may  also 
be  kept  in  sealed  capillaries  in  the  refrigerator.  By  these  methods 
the  virulence  may  be  preserved  for  months.  Rabbits,  mice  and 
young  rats  are  the  most  susceptible  animals. 

The  pneumococcus  is  the  microbic  agent  in  from  80  to  95 
per  cent  of  cases  of  acute  lobar  pneumonia.  It  also  occurs  in 
otitis  media,  mastoiditis,  meningitis,  peritonitis  and  arthritis. 
Its  presence  is  usually  associated  with  a  fibrino-purulent  exudate. 
In  severe  pneumonia  it  is  often  present  in  the  circulating  blood. 

Pneumonia,  or  inflammation  of  the  lungs,  may  be  caused 
by  a  great  variety  of  organisms,  the  tubercle  bacillus,  the  pneu- 
mobacillus  of  Friedlaender,  the  streptococcus,  the  typhoid 
bacillus  and  many  others.  Typical  lobar  pneumonia,  however, 
a  disease  characterized  by  a  definite  sequence  of  pathological 
changes  in  the  lung  and  by  a  rather  typical  clinical  course,  is 
rarely  caused  by  any  organism  other  than  Diplococcus  pneumonia. 
This  is  a  very  frequent  disease  in  adults  and  doubtless  the  most 
frequent  cause  of  death  in  persons  over  50  years  of  age. 

The  nature  of  the  poisons  produced  by  the  pneumococcus 
is  not  definitely  known.  When  killed  by  heat,  the  dead  germ 
substance  is  not  very  toxic.  One  very  remarkable  property  of 
the  organism  is  its  susceptibility  to  the  action  of  bile  and  solutions 
of  bile  salts.  These  cause  a  complete  and  prompt  solution  of 
suspensions  of  pneumococci.  Cole1  has  shown  that  a  powerful 
poison  is  set  free  by  this  disintegration  of  pneumococci,  the 
toxic  action  of  which  resembles  that  seen  in  the  phenomenon 
of  anaphylaxis. 

1  Cole:  Journ.  Exp.  Med.,  1912,  Vol.  XVI,  pp.  644-664. 


260  SPECIFIC   MICRO-ORGANISMS 

It  has  been  possible  to  induce  a  high  degree  of  immunity  in 
horses,  and  the  serum  of  these  animals  is  protective  and  to  some 
extent  curative  in  animal  experiments.  Practically  it  has  as 
yet  no  place  in  the  treatment  of  human  infections  with  the 
pneumococcus. 

Streptococcus  Viridans. — Schottmueller1  has  found  a  strepto- 
coccus, resembling  in  some  respects  the  pneumococcus,  in  the 
blood  of  cases  of  subacute  endocarditis  or  endocarditis  lenta. 
On  the  blood-agar  plates  the  colonies  appear  after  two  to  five 
days  as  opaque  granules  surrounded  by  a  cloudy  but  distinctly 
greenish  zone.  The  organism  is  being  found  very  frequently 
in  cases  of  subacute  endocarditis,2  and  is  apparently  the  specific 
cause  of  this  particular  fairly  well-defined  type  of  endocarditis. 

Streptococcus  Mucosus. — Schottmueller3  has  isolated  a  strep- 
tococcus from  various  purulent  processes,  which  not  only  pos- 
sesses a  mucoid  capsule  in  the  living  body,  but  also  shows  very 
distinct  capsules  in  artificial  culture.  The  size  of  the  cells  is  ex- 
ceedingly variable.  Serum  agar  or  ascitic-fluid  agar  are  necessary 
for  successful  culture. 

Streptococcus  Pyogenes. — Bacteria  were  observed  in  pyemic 
abscesses  by  Rindfleisch  in  1866  and  in  the  following  years  this 
observation  was  confirmed  by  numeTOUs  pathologists.  Klebs 
(1870-71)  recognized  the  u  Micro  sp  or  on  septicum"  as  the  cause  of 
wound  infections  and  the  accompanying  fever,  as  well  as  the 
resulting  pyemia  and  septicemia.  Ogston  (1882)  first  clearly 
distinguished  between  the  chain-form,  streptococcus,  and  the 
grape-form,  staphylococcus,  of  the  pus  cocci,  not  only  on  the 
basis  of  their  grouping  but  also  in  respect  to  the  types  of  inflamma- 
tion with  which  they  are  associated.  Pure  cultures  were  first 
obtained  by  Fehleisen  (1883)  from  erysipelas  (Streptococcus 
erysipelatos)  and  by  Rosenbach  (1884)  from  the  pus  of  wounds 
(Streptococcus  pyogenes).  The  former  produced  typical  erysipe- 
las by  inoculating  the  human  skin  with  his  cultures.  There 

1  Muenchener  med.  Wochenschr.,  1903,  (I),  No.  20,  p.  849. 

2  Major,  Johns  Hopkins  Hasp.  Bull.,  1912,  Vol.  XXIII,  pp.  326-332. 
*Mnench.  med.  Wochenschr.,  1903,  Bd.  L,  S.  849-853;  S.  909-912. 


COCCACE^E     AND     THEIR    PARASITIC    RELATIONSHIPS  261 

is  no  specific  distinction  between  the  streptococci  found  in  ery- 
sipelas and  those  found  in  other  lesions.  The  difference  in  the 
pathological  process  depends  rather  upon  the  portal  of  entry  of 
the  infection,  the  virulence  of  the  microbe  and  the  resistance  of 
the  host. 

Streptococcus  pyogenes  lives  naturally  upon  the  mucous 
membranes,  especially  in  the  pharynx,  nose  and  mouth,  the 
intestine  and  on  the  vaginal  mucosa.  Such  streptococci  found 
in  normal  individuals  are  relatively  non-virulent.  Virulent 
streptococci  occur  in  erysipelatous  lesions  of  the  skin,  in  infected 
wounds,  on  the  inflamed  pharyngeal  mucosa,  and  in  the  lochia, 
uterine  wall  and  in  the  circulating  blood  in  puerperal  fever. 
Streptococci  are  frequently  found  in  pyemic  abscesses,  bacteremia, 
meningitis  and  pneumonia.  It  seems  probable  that  these  virulent 
races  originate  from  the  ordinary  relatively  harmless  parasitic 
forms  in  some  instances,  when  an  opportunity  is  presented  for 
successful  invasion  of  tissues  by  a  lowered  resistance  of  the  host, 
and  that  by  successive  transfer  from  one  susceptible  individual  to 
another  the  virulence  is  still  further  enhanced. 

The  individual  cells  of  a  chain  vary  in  size  from  0.6  to  1.5^ 
and  in  form  from  flattened  disks  to  long  ovals.  The  chains  are 
variable  in  length  and  in  general  the  more  virulent  types  form 
longer  chains  in  broth  cultures.  In  old  cultures  the  cells  are  very 
irregular  in  size,  and  it  was  once  supposed  that  the  larger  spheres 
were  special  resistant  forms,  "arthrospores."  They  are  now 
regarded  as  involution  or  disintegrating  forms.  The  streptococcus 
stains  readily  and  is  Gram-positive. 

Cultures  on  ordinary  media  are  relatively  poorly  developed 
and  of  short  life.  Broth  or  glucose  broth  serves  very  well,  and 
a  few  cultures  in  series  may  be  obtained  on  glycerin  agar  or  glu- 
cose agar.  Loffler's  blood  serum  is  better  than  these.  Serum 
agar,  ascitic-fluid  agar  and  blood  agar  are  the  best  solid  media 
and  ascitic-fluid  broth  is  an  excellent  fluid  medium  for  cultiva- 
tion of  streptococci.  Blood  agar  is  especially  valuable  in  plating 
pus  or  exudates  because  of  the  rather  characteristic  appearance 


262  SPECIFIC  MICRO-ORGANISMS 

of  the  small  colony  surrounded  by  a  very  clear  zone  of  hemolysis 
which  the  streptococcus  produces  on  this  medium.  In  making 
cultures  from  the  blood  in  bacteremia,  plain  agar  previously 
melted  and  cooled  to  45°  C.  is  mixed  with  freshly  drawn  blood 
of  the  patient  and  allowed  to  solidfy  in  a  Petri  dish.  In  other 
cases  naturally  sterile  defibrinated  rabbit's  blood  may  be  used, 
the  technic  of  plating  being  analogous  to  that  described  for  the 
gonococcus.  The  streptococcus  grows  very  slowly  below  20°  C. 
and  poorly  in  ordinary  gelatin,  which  it  does  not  liquefy.  On 
solid  media,  agar  or  serum-agar,  at  37°  C.,  small  round  elevated 
colonies  develop,  0.5  to  i.o  mm.  in  diameter,  and  they  tend  to 
remain  discreet.  In  broth  only  a  slight  cloud  develops,  but 
considerable  granular  deposit  made  up  of  streptococci  is  found 
at  the  bottom  of  the  tube.  Various  carbohydrates  are  fermented 
with  the  production  of  acid  and  without  formation  of  gas,  but 
the  behavior  of  streptococci  toward  these  substances  seems  so 
variable  that  the  attempts  to  utilize  the  fermentative  power  as 
a  basis  for  classifying  the  streptococci  has  not  led  to  wholly  satis- 
factory results.  The  differences  in  fermentative  power  seem  to 
depend  more  upon  vigor  of  growth  than  upon  essential  qualita- 
tive differences  between  the  streptococci  tested.1 

The  streptococcus  is  relatively  very  resistant  to  heat,  at  times 
requiring  one  to  two  hours  heating  at  65°  C.  or  one  hour  at  70° 
C.  in  order  to  insure  sterility,  according  to  V.  Lingelsheim. 
Most  investigators  have  found  60°  C.  for  twenty  minutes  suffi- 
cient. Its  poisons  seem  to  be  chiefly  intracellular  and  set  free 
upon  disintegration  of  the  organisms.  Soluble  poisons  have 
nevertheless  been  found  in  some  cultures. 

Laboratory  animals  are  not  very  susceptible  to  inoculation 
with  streptococci.  White  mice  and  rabbits  are  most  useful, 
and  they  ordinarily  succumb  to  intraperitoneal  injection  of 
virulent  strains. 

The  enormous  importance  of  the  streptococcus  as  a  cause 
of  sickness  and  death  before  the  aseptic  era  is  difficult  to  realize 

1 V.  Lingelsheim  in  Kolle  und  Wassermann,  Handbuch,  1912,  Bd.  IV,  S.  462. 


COCCACE^E    AND     THEIR    PARASITIC    RELATIONSHIPS  263 

at  the  present  time.  Veritable  epidemics  of  streptococcus  in- 
fection in  the  surgical  and  obstetrical  wards  of  hospitals  made 
this  one  of  the  most  dreaded  of  diseases.  Even  to-day  the 
virulent  streptococcus  is  held  in  great  respect  by  many  surgeons, 
and  cases  of  erysipelas  and  other  recognizable  active  streptococcus 
infections  are  commonly  excluded  from  surgical  wards. 

Erysipelas  is  an  acute  febrile  disease  characterized  by  a  local 
redness  and  edema  of  the  skin  which  tends  to  spread  to  contigu- 
ous areas.  In  the  lymph  spaces  beneath  the  epithelium  there  is  a 
collection  of  leukocytes  and  serum,  and  the  streptococci  are  also 
found  here,  especially  at  the  periphery  of  the  reddened  area. 
In  follicular  tonsilitis  and  many  cases  of  pseudo-membranous 
angina  as  well  as  in  the  pharyngitis  of  scarlet  fever,  streptococci 
occur  in  large  numbers,  and  doubtless  bear  a  causal  relation  to 
at  least  a  part  of  the  pathological  process.  In  true  diphtheria, 
streptococci  seem  to  play  rather  frequently  the  role  of  important 
secondary  invaders.  From  the  pharynx  the  streptococcus 
may  gain  access  to  the  middle  ear  and  the  rnastoid  cells,  to  the 
meninges,  to  the  trachea,  bronchi  and  lungs,  setting  up  purulent 
inflammations  in  any  of  these  locations.  It  is  an  important 
secondary  invader  in  pulmonary  tuberculosis.  The  streptococcus 
seems  also  to  cause  enteritis,  particularly  in  infants.  In  the 
puerperium,  streptococci  are  practically  always  present  in  the  lochia. 
In  spite  of  many  attempts  to  differentiate  between  virulent 
and  non-virulent  types  in  this  situation,  it  is  still  impossible  to 
distinguish  them.  Probably  local  conditions  in  the  uterus  as 
well  as  the  general  condition  of  the  pateint  have  much  to  do  in 
determining  her  resistance  to  infection  of  the  uterine  wall  with 
these  normal  streptococci.  Undoubtedly  the  frightful  epidemics 
of  puerperal  fever  in  some  hospitals  previous  to  1875  was  due  to 
the  transference  of  virulent  organisms  from  patient  to  patient 
by  the  attending  physicians  and  nurses.  This  was  first  suggested 
by  Holmes  (1843)  and  more  definitely  proven  by  Semmelweiss 
(1861),  but  their  ideas  received  little  credence  until  the  last 
quarter  of  the  nineteenth  century.  Streptococcus  bacteremia 


264  SPECIFIC   MICRO-ORGANISMS 

is  commonly  a  terminal  phenomenon,  but  it  may  occur  without 
immediate  fatal  issue,  and  may  result  in  endocarditis  and  strepto- 
coccus arthritis. 

Immunity  to  streptococcus  infection  is  slight  in  degree  and 
very  temporary.  Koch  showed  that  erysipelas  could  be  repeat- 
edly produced  on  the  same  area  of  the  skin  by  inoculation  at  inter- 
vals of  10  to  12  days.  Rabbits  and  horses  acquire  a  high  degree 
of  immunity  when  treated  with  gradually  increasing  doses  of 
many  different  strains  of  streptococci.  The  serum  of  such 
animals  has  a  marked  protective  influence  when  injected  into 
animals  and  has  been  employed  in  treating  human  infections, 
in  some  cases  with  success,  while  in  others  the  serum  has  appar- 
ently exerted  no  influence  on  the  course  of  the  disease.  In  local- 
ized chronic  streptococcus  infections,  treatment  with  autogenous 
bacterial  vaccines  (bacteria  suspended  in  salt  solution  and  killed 
by  heat)  seems  to  produce  favorable  effects  in  some  cases. 

Streptococcus  Lacticus  (Micrococcus  Ovalis). — This  is  a 
variety  of  Streptococcus  pyogenes  growing  normally  in  the  intes- 
tine and  of  special  importance  as  the  cause  of  the  normal  souring 
of  milk. 

Staphylococcus  (Micrococcus)  Aureus. — By  the  early  ob- 
servers (Rindfleisch,  Klebs)  this  organism  was  not  distin- 
guished from  the  streptococcus.  Pasteur  in  1880  obtained  it  in 
broth  cultures  from  pus.  Ogston  in  1882  clearly  distinguished  it 
from  the  streptococcus.  Rosenbach  (1884)  by  his  extensive  inves- 
tigations established  the  position  of  the  Staphylococcus  as  a 
cause  of  wound  infection  and  of  osteomyelitis. 

Staphylococci  have  their  natural  habitat  on  the  skin,  in  the 
mouth,  in  the  nasal  cavities  and  in  the  intestine,  without  the 
presence  of  inflammation.  More  virulent  forms  occur  in  in- 
fected wounds,  furuncles,  carbuncles,  various  localized  purulent 
inflammations,  bacteremia  (staphylococcemia),  endocarditis, 
osteomyelitis,  meningitis  and  pneumonia. 

The  cell  is  spherical,  0.7  to  0.9,"  in  diameter.  Division 
takes  place  in  various  planes,  giving  rise  to  irregular  bunches  of 


COCCACE^:    AND     THEIR    PARASITIC    RELATIONSHIPS  265 

cocci.  The  organism  stains  readily  and  is  Gram-positive.  Cul- 
tures are  readily  obtained  on  all  the  common  media  and  growth 
occurs  between  9°  and  42°,  best  at  37°  C.  Broth  is  diffusely 
clouded  with  abundant  sediment.  In  gelatin  stab-culture, 
growth  occurs  all  along  the  line  of  inoculation 
with  funnel-shaped  liquefaction  (Figure  106). 
On  agar  slant  the  growth  is  confluent  and 
yellowish  after  24  hours.  There  is  similar 
growth  on  Loffler's  serum,  often  with  lique- 
faction of  the  medium. 

The  staphylococcus  is  relatively  resistant 
to  heat  and  chemical  germicides.  It  is  killed 
at  62°  C.  in  ten  minutes  and  at  70°  C.  in  five 
minutes.  V.  Lingelsheim1  found  it  more  resis- 
tant, requiring  ten  minutes  at  80°  C.  and  an 
hour  at  70°  C.  to  kill  his  strains,  but  his  fig- 
ures cannot  be  accepted  without  further  con- 
firmation.2 It  is  about  as  resistant  to  chemical 
poisons  as  any  of  the  sporeless  bacteria,  and 
is  commonly  employed  as  a  test  object  in  the 
investigation  of  germicides.  Mercuric  chlo- 
ride i- 1 ooo  requires  three  to  five  hours  to  kill 
staphylococcus  cultures  and  much  longer  if 
the  organisms  are  present  in  pus.  Carbolic  FlG  I06.— Gelatine 
acid,  3  per  cent,  kills  them  in  two  to  ten  culture  staphylococcus 

aureus  one  week  old. 

minutes. 

The  pigment  is  a  lipochrome  and  is  produced  only  in  the 
presence  of  oxygen.  The  tryptic  ferment  diffuses  out  of  the  cells 
and  is  capable  of  liquefying  gelatin,  albumen  and  fibrin.  The 
staphylococcus  produces  a  soluble  poison  which  kills  leukocytes 
(leukocidin)  and  others  which  dissolve  red  blood  cells  (staphy- 
lolysin)  and  cause  clumping  of  red  blood  cells  (agglomerin) . 
These  substances  are  true  soluble  toxins  and  they  are  destroyed 

1  Neisser:  Kolle  und  Wassermann,  Handbuch,  1912,  Bd.  IV,  S.  361. 

2  Compare  with  similar  tests  on  streptococci  by  v.  Lingelsheim,  p.  262. 


266  SPECIFIC   MICRO-ORGANISMS 

by  heating  to  80°  C.  Other  soluble  poisons  seem  also  to  be  pre- 
sent. The  bacterial  cells  killed  by  heat  are  only  slightly  toxic, 
yet  it  is  very  probable  that  in  the  disintegration  of  the  cocci  in 
an  inflammatory  process  more  poisonous  substances  may  be 
derived  from  their  cell  protein. 

Rabbits  are  the  animals  of  choice  for  inoculation  with  staphy- 
lococci.  Intravenous  injection  with  virulent  cultures  usually 
causes  multiple  abscesses  in  the  internal  organs  with  death  in 
4  to  8  days.  Typical  endocarditis  has  been  produced  by  injected 
organisms  from  potato  cultures,  and  with  greater  certainty  when 
the  heart  valves  are  injured  mechanically,  especially  in  young 
rabbits.  Osteomyelitis  sometimes  follows  intravenous  injection 
in  growing  rabbits,  especially  if  the  bone  be  slightly  injured 
at  the  time  of  inoculation.  In  man,  typical  furuncles  and  carbun- 
cles have  been  produced  by  rubbing  pure  cultures  on  the  skin 
(Garre  1885)  and  by  subcutaneous  injection. 

In  man  this  organism  is  a  frequent  cause  of  local  purulent 
inflammations,  and  it  sometimes  gives  rise  to  pyemic  abscesses 
and  general  bacteremia.  Recurrent  furuncles  and  carbuncles 
are  ordinarily  due  to  staphylococci. 

Animals  have  been  immunized  to  staphylococci  but  the  serum 
obtained  from  them  has  relatively  slight  value  in  treatment. 
Specific  treatment  by  means  of  dead  bacterial  cells,  bacterial 
vaccines,  has  been  developed  by  A.  E.  Wright  and  has  proved 
its  value  in  the  treatment  of  chronic  furunculosis.  A  suspension 
in  salt  solution  of  bacterial  cells  from  an  agar  slant,  sterilized 
by  heating  to  60-65°  C.  for  30  minutes  and  standardized  by 
microscopic  count  of  the  bacterial  cells,  is  employed.  Doses 
from  50  million  to  1000  million  bacterial  cells  are  injected  two 
or  three  times  a  week  for  a  long  period  of  time,  the  size  and  fre- 
quency of  dosage  being  governed  by  the  clinical  condition  of  the 
patient.  Determination  of  the  opsonic  index  is  probably  un- 
necessary and  is  now  quite  generally  neglected.  Autogenous 
vaccines  (made  with  the  staphylococcus  isolated  from  the  patient) 
are  usually  superior  to  stock  vaccines. 


COCCACE.E     AND     THEIR     PARASITIC    RELATIONSHIPS  267 

Staphylococcus  Albus. — This  is  quite  similar  to  Staphylococcus 
aureus  in  all  respects  except  pigment  production.  Usually, 
but  not  always  it  is  less  virulent.  Staph.  epidermidis  (Welch)  is 
an  avirulent  variety  of  Staph.  albus,  very  abundant  on  the  normal 
skin.  Many  other  varieties  of  staphylococci  have  been  described. 

Micrococcus  Tetragenus. — This  organism  occurs  in  lung 
cavities  in  phthisis,  and  in  the  sputum,  usually  in  groups  of  four 
cells,  tetrads,  enclosed  in  a  transparent  capsule.  It  is  Gram- 
positive,  grows  on  ordinary  media  and  does  not  liquefy  gelatin. 
White  mice  and  guinea-pigs  are  susceptible  and  ordinarily  die 
of  general  bacteremia  in  two  to  six  days  after  inoculation.  The 
pathogenic  role  of  the  organism  in  man  is  doubtful. 

Sarcina  Ventriculi. — Goodsir  in  1842  observed  sarcines  in 
vomitus.  The  coccus  is  large,  2.5^  in  diameter,  and  occurs  in 
cubes  of  eight  cells  or  as  large  conglomerates  of  these.  It  grows 
on  ordinary  media,  usually  producing  a  yellow  pigment.  It 
is  found  in  the  stomach  in  some  conditions  in  which  the  acidity 
of  the  gastric  juice  is  diminished.  It  is  apparently  non-pathogenic. 

Sarcina  Aurantiaca. — This  is  a  common  saprophytic  coccus 
found  in  fermenting  liquids  and  occasionally  in  the  air.  It 
grows  well  on  ordinary  media  and  liquefies  gelatin.  An  orange 
pigment  is  produced.  Typical  packets  are  produced  in  liquid 
media,  especially  in  hay  infusions. 

Micrococcus  (Planococcus)  Agilis. — This  organism  occurs 
in  surface  waters.  It  liquefies  gelatin  and  produces  a  rose-red 
pigment  on  agar  and  potato.  Its  remarkable  feature  is  the 
possession  of  a  flagellum  and  active  motility.  It  is  Gram-positive. 


CHAPTER  XVII. 
BACTERIACE^E:  THE  SPOROGENIC  AEROBES. 

The  aerobic  spore-forming  bacilli  are  essentially  inhabitants 
of  the  soil  and  the  fermenting  organic  material  likely  to  occur 
there.  Along  with  a  few  species  of  this*  group  we  shall  consider 
one  pathogenic  sporogenous  bacterium,  the  anthrax  bacillus, 
which  resembles  them  very  closely  except  in  its  virulence  for 
animals  and  its  lack  of  active  motion,  both  of  which  may  perhaps 
justly  be  regarded  as  variations  from  the  group  type  due  to  its 
parasitic  mode  of  life. 

Bacillus  Mycoides. — This  organism  is  universally  distributed 
in  fertile  soils  and  also  occurs  in  surface  waters  and  in  the  air. 
It  is  a  large  rod  with  rounded  ends,  usually  growing  in  threads. 
Large  median  spores  are  formed  without  distorting  the  cell. 
It  is  motile  but  rather  sluggish.  Growth  occurs  on  all  ordinary 
media.  In  gelatin  stab-culture,  thread-like  processes  extend 
out  on  all  sides  from  the  line  of  puncture  giving  the  appearance 
of  an  inverted  pine  tree.  Later  the  gelatin  becomes  entirely 
liquefied.  The  organism  is  an  important  agent  in  the  decompo- 
sition of  plant  residues  in  the  soil.  It  is  without  pathogenic 
properties. 

Bacillus  (Mesentericus)  Vulgatus. — This  is  another  widely 
distributed  soil  bacterium.  It  is  commonly  called  the  potato 
bacillus.  The  cell  is  short  and  relatively  thick  with  rounded 
ends,  actively  motile,  often  in  pairs  or  threads.  Large  spherical 
median  spores  are  produced  without  distortion  of  the  cell. 
These  spores  are  very  resistant  to  heat  and  germicides,  sometimes 
surviving  the  temperature  of  boiling  water  for  several  hours. 
B.  vulgatus  grows  well  on  all  ordinary  media.  Gelatin  is  liquefied. 
Milk  is  coagulated  and  then  digested.  On  potato  a  wrinkled 

268 


BACTERIACE^E :  THE  SPOROGENIC  AEROBES 


269 


membrane  is  produced,  so  characteristic  that  the  name  "mesen- 
tericus"  was  applied  to  this  species.     It  is  not  pathogenic. 

Bacillus  Subtilis. — Bacillus  subtilis,  or  the  hay  bacillus,  is 
abundant  in  the  soil  and  on  the  surface  of  plants,  and  common 
in  surface  waters  and  in  the  air.  It  is  readily  obtained  by  boiling 
hay  in  water  and  then  setting  the  infusion  aside  for  a  few  days. 
The  cell  is  relatively  large,  about  1.2,"  wide  by  5^  long,  with 
ends  somewhat  rounded.  Long  threads  are  commonly  formed. 
It  is  motile  with  peritrichous  flagella.  Large  oval  median  spores 


FIG.   107. — Bacillus  subtilis.     Xiooo. 

are  formed  without  distortion  of  the  cell  and  these  are  almost 
as  resistant  as  the  spores  of  the  potato  bacillus.  B.  subtilis 
grows  rapidly  on  ordinary  media  in  the  presence  of  air,  best  at 
about  30°  C.  Gelatin  is  liquefied  and  milk  is  digested.  The 
organism  is  typically  saprophytic,  but  it  has  been  found  growing 
in  the  intestine  by  some  investigators,  and  has  been  found  in  a 
few  instances  in  infections  of  the  human  eye,  cases  of  pan- 
ophthalmitis  following  injury.1 

1  Silber schmidt:  Annales   de   V I nstitut  Pasteur,  1903,  Vol.  XVII,  pp.  268-287; 
Also  see  Kneass  and  Sailer:  Univ.  Penn.  Med.  Bull.,  June,  1903,  Vol.  XVI,  pp. 


270  SPECIFIC  MICRO-ORGANISMS 

Bacillus  (Bacterium)  Anthracis. — Pollender  in  1849  and 
Davaine  and  Rayer  in  1850  observed  thread-like  bodies  in  the 
blood  of  animals  dying  of  anthrax.  Robert  Koch  in  1876  obtained 
pure  cultures  of  the  organism,  using  the  aqueous  humor  of  the 
ox's  eye  as  culture  medium.  He  saw  the  small  rod-shaped  bodies 
found  in  the  anthrax  blood  elongate  into  threads  in  this  medium, 
and  observed  the  formation  of  the  bright  refractive  bodies  in 
these  threads,  which  he  correctly  recognized  as  spores.  Finally 
by  inoculating  healthy  animals  with  his  cultures  he  produced 


FIG.  108. — Anthrax  bacilli  in  the  capillaries  of  the  liver  of  a  mouse. 

typical  anthrax  in  them,  thus  proving  conclusively  for  the  first 
time  the  causal  relation  of  a  bacterium  to  a  disease. 

The  anthrax  bacillus  occurs  in  the  blood  and  throughout  the 
tissues  of  animals  suffering  from  anthrax,  and  in  the  excretions 
of  such  animals.  Its  spores  occur  on  hides  and  in  wool  derived 
from  anthrax  animals.  Furthermore,  the  soil  of  fields  where 
anthrax  animals  have  grazed  harbors  these  organisms  for  many 
years.  It  seems  probable  that  the  bacilli  multiply  in  the  soil 
during  the  warm  wet  seasons  and  it  is  certain  that  the  spores 
may  lie  dormant  for  as  long  as  ten  years  in  dry  places. 


BACTERIAC.E:  THE  SPOROGENIC  AEROBES 


271 


The  cell  is  about  1.25^  wide  and  3  to  io,«  long,  with  rounded 
ends  when  single,  but  in   the  threads  the  contiguous  ends  are 


.;•%     .  *  V***    •/•••*- 

" '        ' 


FIG.  109. — Bact.  anthracis.     Spore  production.     (From  Marshall  after  Migula.} 

square-cut.  In  the  circulating  blood  the  bacilli  are  single  or  in 
pairs  and  spores  are  never  formed  in  the  animal  body  (Fig.  108). 
In  cultures  long  threads  are  produced  and  spores  are  usually 


FIG.  no. — Bact.  anthracis.     Colony  upon  a  gelatin  plate.     Xioo.     (After  Fraenkel 

and  Pfei/er.) 

formed  after  24  to  48  hours  (Fig.  109).     The  anthrax  bacillus 
is  aerobic  and  grows  readily  on  all  ordinary  media,  best  at  37°  C. 


272  SPECIFIC   MICRO-ORGANISMS 

Gelatin  is  slowly  liquefied.  The  colony  presents  a  very  char- 
acteristic appearance,  especially  as  it  grows  on  gelatin,  which  is 
due  to  the  large  coils  of  long  parallel  threads,  of  which  the  colony 
is  composed.  The  vegetative  bacillus  is  rather  easily  killed  but 
the  spores  may  survive  boiling  in  water  for  5  minutes  and  in 
some  instances  as  long  as  half  an  hour  when  afforded  some 
mechanical  protection.  Chemical  germicides  cannot  be  relied 
upon  to  destroy  the  spores.  Sterilization  in  the  autoclave  is 
the  safest  method  of  disposing  of  anthrax  material. 

Anthrax  is  a  disease  which  occurs  spontaneously  in  cattle  and 


FIG.  in. — Bact.  anthracis.     Showing  the  thread  formation  of  colony.     (After  Kolle 

and  Wassermann.) 

sheep  and  rarely  in  horses,  swine  and  in  man.  The  disease  is 
produced  by  inoculation  in  many  other  animals.  Mice,  guinea- 
pigs  and  rabbits  are  susceptible  in  the  order  named.  The  disease 
is  common  in  European  and  Asiatic  stock-raising  districts  and 
in  Argentine  Republic.  Several  local  epizootics  have  occurred  in 
the  United  States  and  a  few  cases  of  human  anthrax.  Experi- 
mental anthrax  is  readily  produced  in  susceptible  animals  by 
subcutaneous  inoculation,  less  certainly  by  feeding  the  spores. 
In  the  acute  form  the  bacilli  are  found  in  large  numbers  every- 
where in  the  blood,  and  this  is  the  common  picture  in  cattle, 
sheep,  rabbits,  guinea-pigs  and  mice.  Chronic  forms  occur, 


BACTERIACE^::  THE  SPOROGENIC  AEROBES        273 

however,  either  because  of  lowered  virulence  of  the  germ  or  of 
increased  resistance  of  the  host,  and  in  these  cases  the  bacteria 
may  be  very  scarce  and  difficult  to  find  microscopically,  even 
after  death  of  the  animal.  Cultures  from  the  spleen  will  usually 
show  the  presence  of  the  bacillus  there.  The  mechanism  by 
which  the  bacillus  causes  death  is  unknown.  In  the  acute  cases, 
as  in  the  mouse,  the  bacilli  are  so  abundant  in  the  blood  that 
mechanical  interference  with  the  circulation  seems  a  plausible 
explanation,  but  this  certainly  does  not  suffice  for  other  types  of 
the  disease  in  which  chemical  poisoning  must  play  the  chief 
role.  So  far  it  has  not  been  possible  to  demonstrate  any  powerful 
poisons  in  cultures  of  the  anthrax  bacillus.  It  is  probable  that 
the  essential  poisons  are  produced  by  a  reaction  between  the 
substance  of  the  bacillus  and  the  fluids  of  the  host,  particularly 
the  enzymes  of  the  latter,  which  cause  disintegration  of  the  bac- 
terial bodies. 

The  infection  is  acquired  by  grazing  animals  through  the 
alimentary  tract  primarily,  but  also  to  some  extent  by  inoculation 
(contact,  flies,  intermediate  objects).  In  man  there  are  three 
recognized  types  (a)  malignant  pustule,  (b)  pulmonary  anthrax, 
and  (c)  intestinal  anthrax.  Malignant  pustule  results  from  in- 
oculation of  the  skin,  especially  in  those  who  handle  hides  or  care 
for  anthrax  animals.  It  is  at  first  a  local  pustular  and  necrotic 
lesion  tending  to  involve  contiguous  tissue  by  extension,  but  soon 
invading  the  lymph  vessels  and  walls  of  the  veins.  The  bacteria 
thus  gain  the  blood  stream  and  a  rapidly  fatal  general  bacteremia 
supervenes.  Recovery  sometimes  occurs  before  the  disease  be- 
comes generalized.  Pulmonary  anthrax  is  caused  by  inhalation 
of  anthrax  spores  (woolsorter's  disease).  Intestinal  anthrax 
is  uncommon  in  man  but  has  occurred.  Both  are  very  fatal 
forms  of  the  disease. 

Immunity  to  anthrax  was  first  successfully  produced  by  Pas- 
teur through  vaccination  with  attenuated  living  cultures.  Broth 
cultures  inoculated  with  bacilli  taken  directly  from  the  animal 
body  were  grown  at  42°C  to  43°C.  At  this  temperature  spores 

18 


274  SPECIFIC   MICRO-ORGANISMS 

are  not  produced  and  the  bacillus  gradually  loses  its  virulence. 
When  it  will  no  longer  kill  guinea-pigs  but  will  still  kill  mice  the 
strain  is  again  grown  at  37°C.  and  injected  into  cattle  and  sheep 
as  the  first  vaccine.  Twelve  days  later  a  second  vaccine  is  in- 
jected, which  is  a  somewhat  more  virulent  culture,  still  capable 
of  killing  guinea-pigs  but  not  powerful  enough  to  cause  fatal  in- 
fection of  rabbits.  As  a  result  of  these  two  treatments,  nearly 
all  animals  become  immune  to  the  natural  disease  or  to  inocula- 
tion with  fully  virulent  cultures.  Sobernheim1  and  Sclavo2  have 
induced  a  high  degree  of  immunity  in  sheep  and  in  asses  by  re- 
peated injections  of  the  bacilli,  and  have  found  the  serum  of  such 
hyper-immune  animals  to  be  protective  and  curative  upon  in- 
jection into  other  animals.  The  injection  of  this  serum  along 
with  a  dose  of  living  culture  of  about  the  strength  of  Pasteur's 
second  vaccine  has  been  employed  in  immunizing  cattle  and 
sheep.  All  the  necessary  treatment  is  thus  given  at  one  time. 
The  serum  has  also  been  successfully  employed  in  conjunction 
with  the  appropriate  medical  and  surgical  measures  in  the  treat- 
ment of  malignant  pustule  in  man.3 

Sobernheim:   Zeitsch.  f.  Hyg.,  1897,  XXV,  pp.  301-356;  Centralbl.  f.  Bakt., 
1899,  XXV,  p.  840. 

2  Sclavo:  Centralbl.  f.  Bakt.,  1899,  XXVI,  p.  425. 

3  For  a  discussion  of  treatment  of  human  anthrax  consult  Boidin,  Vignaud  and 
Fortineau,  Presse  Medicale,  Aug.  14,  1912;  also  Becker,  Munch,  med.  Wochenschr., 
Jan.  23,  1912. 


CHAPTER  XVIII. 
BACTERIACE^:  THE  SPOROGENIC  ANAEROBES. 

The  bacteria  of  this  group  are  hindered  in  their  development 
by  the  presence  of  free  oxygen  and  their  artificial  culture  is  ordi- 
narily successful  only  when  they  are  protected  from  oxygen,  at 
least  in  the  early  stages  of  development.  Like  the  sporogenic 
aerobes,  they  live  in  the  soil,  but  they  are  associated  here  more 
especially  with  decomposing  materials  of  animal  origin,  and  are 
less  frequently  found  in  soils  which  have  not  received  fertilizers 
from  animal  sources.  There  is  good  reason  to  believe  that  their 
essential  habitat  is  the  intestinal  canal  of  animals,  especially  the 
mammals,  and  that  their  life  in  the  soil  does  not  represent  the 
most  active  stage  of  their  existence,  but  that  they  reach  the  soil 
with  animal  excreta  and  the  bodies  of  dead  animals  and  continue 
to  live  in  the  soil  for  a  considerable  period. 

Bacillus  Edematis. — Pasteur  in  1877  injected  infusions  of 
putrid  flesh  into  laboratory  animals  and  produced  a  fatal  sub- 
cutaneous edema  with  penetration  of  the  bacteria  into  the  blood 
in  some  instances.  The  organism  which  he  called  "Vibrion 
septique"  was  found  to  be  an  obligate  anaerobe,  the  first  anae- 
robic organism  ever  recognized.  Koch  (1881)  studied  the  organism 
in  pure  culture  on  solid  media  and  named  it  Bacillus  edematis 
maligni.  The  recognized  type  organism  is  that  studied  by 
Koch. 

The  bacillus  is  very  widely  distributed  in  soil  and  dust,  and 
is  very  common  in  the  feces  of  herbivorous  animals.  It  is  es- 
pecially abundant  in  putrefying  animal  matter.  The  cell  is  about 
i/x  thick  by  3/i  in  length,  although  considerable  variation  in  size 
and  shape  occurs.  It  is  usually  slightly  motile  and  possesses 
peritrichous  flagella,  stains  readily,  is  only  relatively  Gram-posi- 

275 


276  SPECIFIC  MICRO-ORGANISMS 

tive,  some  of  the  cells  being  decolorized  by  prolonged  treatment 
with  alcohol.  The  spores  are  central,  or  intermediate  in  position 
with  bulging  of  the  cell. 

In  cultures  B.  edematis  is  a  strict  anaerobe.  It  liquefies  gela- 
tin. Milk  is  slowly  coagulated  and  the  coagulum  digested,  the 
reaction  remaining  alkaline  to  litmus.  The  cultures  have  a  foul 
odor.  The  spores  withstand  boiling  sometimes  for  2  to  3  hours. 
The  morphological  and  physiological  properties  of  this  organism 
are  quite  variable  and  the  many  intermediate  types  between  it 
and  B.  feseri  make  distinction  between  the  two  species  somewhat 
difficult. 

In  animals  and  man,  malignant  edema  occurs  spontaneously 
as  a  wound  infection,  but  it  is  not  very  common.  It  has  been 
observed  most  frequently  in  horses  and  in  new-born  calves.  The 
guinea-pig  is  susceptible.  In  general  a  mere  injection  of  the 
bacilli  fails  to  produce  serious  disease.  The  presence  of  foreign 
bodies  or  extensive  tissue  destruction  favors  the  infection. 

Bacillus  Feseri. — Feser  and  Bollinger  (1875-1878)  observed 
the  large  narrow  rods  in  the  diseased  tissues  and  exudates  of 
symptomatic  anthrax  or  black  leg,  a  fatal  disease  of  cattle  and 
sheep.  Man  is  not  affected.  Arloing,  Cornevin  and  Thomas 
(1884)  obtained  the  organism  in  culture.  The  organism  is  a 
strict  anaerobe  and  resembles  B.  edematis  very  closely.  Black 
leg  is  a  local  emphysematous  inflammation  usually  beginning  in 
one  leg  of  cattle  or  sheep,  rapidly  extending  and  resulting  in  death 
as  a  rule.  Immunity  is  obtained  by  injecting  small  doses  of  the 
virulent  bacteria  or  by  injecting  attenuated  organisms,  and  also 
by  injecting  the  virus  together  with  an  immune  serum.1 

B.  Welchii. — Welch  and  Nuttall  in  1892  discovered  this  organ- 
ism at  autopsy  in  a  body  showing  general  emphysema  of  the 
tissues  and  gas  bubbles  in  the  blood-vessels.  They  obtained 
cultures  by  anaerobic  methods  and  caused  similar  post-mortem 
emphysema  in  the  bodies  of  rabbits.  The  organism  lives  and 
multiplies  in  the  intestine  of  man  and  other  mammals,  is  widely 
1  Kitt,  Kolle  and  Wassermann,  Handbuch,  1912,  Bd.  IV,  S.  819-836. 


BACTERIACE^E :    THE    SPOROGENIC   ANAEROBES  277 

distributed  in  the  soil  and  is  commonly  present  in  milk  and  other 
animal  food  products.  The  cell  is  a  large  rod  surrounded  by_a 
capsule  when  grown  on  media  rich  in  protein  or  in  the  animal 
body.  The  width  of  the  cell  (without  capsule)  varies1  from  i.i  to 
i.fu  with  a  mean  of  1.3^  and  the  length  from  2.6  to  7.6/1,  with  an 
average  of  4.6/z,  the  measurements  being  made  on  organisms 
grown  in  an  agar  stab-culture  24  hours  at  37°  C.  When  grown 
in  blood  broth  the  germ  is  capsulated  and  the  measurements,  in- 
cluding the  capsule  are  as  follows:  width  1.9  to  2.5/1  with  average 
of  2.i;u,  and  length,  2.8  to  6. 6 /*  with  average  of  4.7/11.  Usually  the 
organism  is  non-motile,  but  flagella  can  sometimes  be  demon- 
strated. In  the  intestine  and  in  protein  media  the  organism 
forms  spores,  usually  median  without  bulging  of  the  cell,  but 
these  are  not  commonly  observed  in  cultures.  The  organism  is 
a  strict  anaerobe.  Its  most  striking  property  is  the  enormously 
rapid  production  of  gas  in  media  containing  dextrose  or  lactose. 
Cultures  are  obtained  most  readily  by  heating  a  suspension  of 
feces  to  80°  C.  for  15  minutes  and  inoculating  it  into  glucose  broth 
mixed  with  blood  in  a  Smith  fermentation  tube.  After  24  to  48 
hours  incubation  its  presence  will  usually  be  revealed  by  abun- 
dant production  of  gas.  Milk  is  coagulated  and  rendered  acid 
with  an  abundant  production  of  gas  (stormy  fermentation). 
On  blood-agar  plates  incubated  in  hydrogen,  the  colony  is  round 
with  regular  outline  and  surrounded  by  a  clear  zone  of  hemolysis. 

Emphysematous  gangrene  occurs  in  man  as  a  rapidly  extend- 
ing, very  fatal  disease,  due  to  the  infection  of  wounds  with  this 
organism.  The  presence  of  necrotic  tissue  seems  to  be  necessary 
in  order  that  the  organism  may  gain  a  foothold,  but  when  once 
begun  the  inflammation  may  extend  with  great  rapidity.  The 
gas  found  in  bodies  at  autopsy  is  usually  the  result  of  an  agonal 
or  a  post-mortem  invasion  by  the  bacilli  from  the  intestine. 

There  are  several  other  types  of  sporogenic  anaerobes  of  the 
same  general  nature  as  B.  edematis,  B.  feseri,  and  B.  welchii,  iso- 

1  The  measurements  are  taken  from  Kerr,  The  Bacillus  welchii,  Thesis,  Univ. 
of  Illinois,  1909. 


278 


SPECIFIC   MICRO-ORGANISMS 


e  soil,  from  the  feces  or  from 


lated  by  various  workers  froi 

putrefying  material. 

Bacillus  Tetani. — Tetanus  has 
been  recognized  as  a  complication,.^ 
wounds  since  the  time  of  Hippocrates/, 
Forscher,  Carle  and  Rattone,  in  1884,) 
first  proved  it  to  be  inoculable  by  in- 
jecting pus  from  a  human  case  into  1 2 
rabbits,  of  which  n  died  of  tetanus. 
Nicolaier  in  1884  produced  tetanus  by 
injecting  soil  into  mice,  guinea-pigs  and 
rabbits,  and  found  a  slender  bacillus  in 
the  animals  at  the  point  of  inoculation. 
He  was  able  to  propagate  the  bacillus 
in  mixed  culture  on  coagulated  sheep's 
serum.  Kitasato  obtained  the  first 
pure  cultures  by  subjecting  the  mixed 
culture  to  a  temperature  of  80°  C.  for 
an  hour,  inoculating  agar  plates  and 
incubating  them  in  an  atmosphere  of 
hydrogen.  With  his  pure  cultures,  he 
caused  typical  tetanus  in  animals. 

The  organism  occurs  in  the  soil 
which  has  received  animal  fertilizers 
and  in  the  intestine  of  herbivorous 
mammals.  The  bacterial  cell  is  0.3  to 
0.5^1  wide  and  2  to  4/4  long,  single  in 
young  cultures,  but  ,often  joined  end 
to  end  to  form  long  threads  in  older 
cultures.  It  is  motile  and  possesses 
abundant  peritrichous  flagella.  The 
spore  is  very  characteristic.  It  is  usu- 
ally spherical,  i  to  1.5  ^  in  diameter, 

FIG.  ii2.— B.     welchii   in    agar 

culture,  showing  gas  formation,  situated  at  the  extremity  of  the  cell, 
giving  it  the  appearance  of  a  drumstick.  The  bacillus  stains 
readily  and  is  Gram-positive, 


BACTERIACE.E :    THE    SPIROGENIC   ANAEROBES  279 

Isolation  of  B.  tetani  from  mixed  material  or  from  wounds 
known  to  contain  it  is  not  always  easy.  The  material  should 
be  planted  in  glucose  broth  and  incubated  in  hydrogen  at  37°  C7 
for  2  to  3  days.  Microscopic  examination  of  the  sediment  may 
then  reveal  the  drumsticks.  Kitasato's  procedure  should  then  be 
followed,  employing  agar  distinctly  alkaline  to  litmus  and  con- 
taining 2  per  cent  of  glucose.  If  many  other  spore-forming  bac- 
teria are  present  in  the  mixture,  special  procedures  are  necessary, 
such  as  preliminary  culture  for  8  days  at  37°  C.  in  a  deep  stab  in 
coagulated  rabbit's  blood  with  subsequent  heating  to  80°  C.  to 
get  rid  of  B.  edematis,  or  culture  for  8  days  at  37°  C.^ih  milk  with 
subsequent  heating  to  get  rid  of  B.  welchii.  Aerobic  spore-formers 
may  be  eliminated  by  successive  transfers  in  animals. 

The  spores  of  B.  tetanic esist  the  temperature  of  boiling  water 
for  5  to  30  minutes.  Biological  products  to  be  introduced  into 
the  human  body  need  to  be  sterilized  in  the  autoclave  or  else 
carefully  examined  by  anaerobic  culture  methods  to  insure  their 
freedom  from  tetanus  spores.  The  danger  of  infection  from  this 
source  has  been  emphasized  by  Smith.1 

The  colony  in  glucose  gelatin  or  glucose  agar  consists  of  a 
compact  center  with  slender,  radiating,  straight  or  irregularly 
curved  threads  about  the  periphery.  Liquefaction  of  gelatin 
becomes  evident  in  stab-culture  after  about  two  weeks  at  20°  C. 
Milk  is  sometimes  but  not  always  coagulated  and  the  casein  is 
eventually  digested. 

The  cultures  of  the  tetanus  bacillus  are  extremely  poisonous, 
especially  so  when  they  are  developed  under  very  strict  anaerobic 
conditions.  A  nerve  poison,  tetanospasmin,  and  a  hemolytic 
poison,  tetanolysin,  are  present.  The  former  is  the  more  impor- 
tant constituent  of  the  tetanus  toxin.  Neutral  or  slightly  alka- 
line plain  nutrient  broth,  incubated  in  an  atmosphere  of  hydrogen 
for  ten  days  after  inoculation  gives  the  most  powerful  toxin. 
The  bacteria-free  fluid  from  such  a  culture  has  been  found  to  kill 
a  mouse  of  ic-grams  weight  in  a  dose  of  o.ooo  005  c.c.  The  toxin  is 

1  Journ.  A.  M.  A.,  Mar.  21,  1908,  Vol.  L.,  pp.  929-934. 


280 


SPECIFIC   MICRO-ORGANISMS 


unstable  in  solution  but  very  stable  when  dried.  Dry  material 
of  which  o.ooo  ooo  i  gram  is  the  fatal  dose  for  a  mouse  is  readily 
obtained.  The  watery  solution  loses  it  toxicity  when  heated  to 
60°  C.  for  20  minutes,  but  when  dry  the  toxin  withstands 
heating  at  120°  C.  for  an  hour. 

Tetanus  presents  essentially  the  same  picture  in  inoculated 
animals  as  in  the  natural  disease,  which  is  indeed,  as  a  general 
rule,  merely  an  accidental  inoculation.  The  presence  of  insoluble 
material  and  of  other  bacteria  mixed  with  them  in  a  wound  favors 
the  development  of  tetanus  bacilli.  The  tetanus  bacilli  always 


FIG.  1 13. — Tetanus  bacilli  showing  terminal  spores.     (After  Kolle  and  Wassermann.} 

remain  localized  near  the  point  of  inoculation  and  may  be  hard 
to  find.  The  poison  produced  by  the  organisms  is  probably  ab- 
sorbed by  the  nerve  endings1  and  transmitted  to  the  central  nerv- 
ous system  through  the  axis  cylinders  or  in  the  perineural  lymph 
spaces  of  the  motor  neurones  rather  than  through  the  blood 
stream.  The  symptoms  arise  after  the  poison  reaches  the  central 
nervous  system  in  sufficient  concentration  to  stimulate  the  nerve 
cells.  In  guinea-pigs  and  mice  the  spasm  always  begins  near  the 
point  of  inoculation,  but  in  man  and  the  large  mammals  it  often 
begins  in  the  muscles  of  the  jaw  and  neck  regardless  of  the  location 

1  Von  Lingelsheim,  Kolle  and  Wassermann,  Handbuch,  1912,  Bd.  IV,  S.  766. 


BACTERIACE.E  I   THE    SPOROGENIC   ANAEROBES 


28l 


of  the  wound.  Wassermann  and  Takaki  have  shown  that  o.i 
gram  of  brain  substance  suspended  in  salt  solution  is  able  to  neu- 
tralize 10  fatal  doses  of  tetanus  toxin,  forming  a  loose  combina- 
tion from  which  the  toxin  may  be  set 
free  by  drying.  Most  mammals  are 
very  susceptible,  although  cats  and 
dogs  are  only  slightly  so.  Birds  are 
relatively  resistant  and  some  reptiles 
are  wholly  refractory  to  the  tetanus 
toxin. 

Von  Behring  and  Kitasato  in  1890 
produced  immunity  in  rabbits,  and 
later  in  horses,  by  injecting  into  them 
toxin  to  which  iodine  trichloride  had 
been  added,  and  subsequently  unal- 
tered toxin.  The  immunized  animal 
was  able  to  survive  an  injection  many 
times  greater  than  the  amount  neces- 
sary to  kill  a  normal  animal.  More- 
over, the  cell-free  blood  serum  of  the 
immunized  animal  was  found  to  neu- 
tralize the  poison  in  a  test-tube  and 
to  protect  a  normal  animal  against 
fatal  doses  of  it.  The  new  substance 
of  the  blood  capable  of  rendering  the 
toxin  harmless  was  called  antitoxin. 
One  antitoxic  unit  of  tetanus  anti- 

, .  i  T         -r»   i     •          •       after  Fraenkel  and  Pfeiffer.) 

toxin,  according   to   Von  Behring,  is 

the  amount  which  will  neutralize  40  million  times  the  amount 
of  fresh  tetanus  toxin  necessary  to  kill  a  mouse  weighing  15 
grams  (40  million  X  the  15  +  Ms  dose)  so  completely  that 
only  a  slight  local  contraction,  indicated  by  a  folding  of  the 
skin,  results  from  subcutaneous  injection  of  the  mixture  into  a 
mouse  (the  L0  effect).  This  amount  of  toxin  (40  million  X  the 
15  +  Ms  dose)  is  generally  measured  in  practice  against  a  standard 


FIG.  114. — Bacillus  tetani. 
Stab  culture  in  glucose  gelatin, 
six  days  old.  (From  McFarland 


282  SPECIFIC   MICRO-ORGANISMS 

antitoxin  and  is  designated  as  a  toxic  unit.  The  toxin  is  pre- 
served in  a  dry  state.  To  test  a  new  antitoxin  one  employs 
ToVo-  of  a  toxic  unit  (40,  ooo  X  the  15  +  Ms  dose)  and  ascertains 
the  amount  of  serum  which  must  be  added  so  as  to  neutralize  it 
to  the  L0  end  point.  Each  trial  mixture  is  diluted  to  i  c.c.  with 
salt  solution  and  0.25  c.c.  per  10  grams  of  body  weight  is  injected 
into  a  mouse.  When  the  typical  L0  effect  is  produced  in  the 
mouse,  the  amount  of  antitoxic  serum  employed  in  the  prepara- 
tion of  this  particular  mixture  is  said  to  represent  ToW  anti- 
toxic unit.  Ordinarily  the  mixtutre  of  toxin  and  antitoxin  is 


A  <  -\ 


FIG.  115. — Bacillus    botulinus.     Some   individuals    containing  spores.     (After  van 

Ermengem.) 

allowed  to  stand  30  minutes  before  injection.  Comparable  re- 
sults are  obtained  only  by  following  a  definite  procedure  and  it 
is  especially  necessary  to  use  the  conventional  dose  of  roW 
antitoxic  unit  and  -nrVo  toxic  unit  in  the  standardization  of 
sera. 

The  standard  unit  employed  in  the  United  States  is  some- 
what different  from  the  Von  Behring  antitoxic  unit.  The  Ameri- 
can immunity  unit  of  tetanus  antitoxin  is  ten  times  the  least 
amount  of  antitetanic  serum  necessary  to  preserve  the  life  of  a 
guinea-pig  weighing  350  grams  for  96  hours  against  the  official 


BACTERIACE^E :   THE   SPOROGENIC   ANAEROBES  283 

test  dose  of  standard  tetanus  toxin  furnished  by  the  Hygienic 
Laboratory  of  the  U.  S.  Public  Health  Service.1  Tetanus  anti- 
toxin deteriorates  with  moderate  rapidity.  The  reaction  be- 
tween tetanus  toxin  and  antitoxin  seems  to  take  place  in  two 
stages,  first  a  reversible  absorption  and  following  this  a  specific 
chemical  union. 

Tetanus  antitoxin  seems  to  be  an  absolute  preventive  of  teta- 
nus if  given  soon  after  the  wound  is  inflicted  in  a  dose  of  20  anti- 
toxic units  (German)  or  1500  immunity  units  (U.  S.  Standard). 
After  symptoms  of  tetanus  have  appeared,  antitoxin  is  of  less 
use.  At  this  time  the  poison  is  present  not  only  in  the  vicinity 
of  the  wound  and  in  the  blood  but  also  in  the  peripheral  nerves 
and  in  the  central  nervous  system.  The  toxin  in  the  last  two  situ- 
ations is  only  slightly  or  not  at  all  influenced  by  subcutaneous  in- 
jection of  antitoxin.  That  in  the  peripheral  nerves  may  be  reached 
by  intraneural  injection,  and  in  subacute  or  chronic  cases  recovery 
may  sometimes  take  place.  Acute  cases  in  which  symptoms 
appear  in  a  few  days  after  infliction  of  the  wound  offer  no  hope. 
Prophylactic  use  of  tetanus  antitoxin  in  all  punctured  and  lacer- 
ated wounds,  especially  those  caused  by  gunpowder  (Fourth  of 
July)  is  an  essential  feature  of  the  effective  treatment  for  tet- 
anus. Surgical  cleansing  and  antiseptic  open  treatment  of  such 
wounds  is  to  be  recommended.2 

s 

Bacillus  Botulinus, — Van  Ermengem  in  1895  discovered  the 
spores  of  this  organism  in  the  intermuscular  connective  tissue 
of  a  ham  which  had  given  rise  to  30  cases  of  food  poisoning  with 
3  deaths.  Other  anaerobic  as  well  as  aerobic  bacteria  were  also 
present  in  the  meat.  Its  natural  habitat  is  unknown  but  it  seems 
to  occur  in  the  feces  of  swine.  The  bacillus  is  0.9  to  i.2/*  wide 
by  4  to  6/z  long  and  occurs  single  or  in  pairs.  It  is  slightly 
motile  and  has  4  to  8  peritrichous  flagella.  It  is  Gram-positive. 
The  spores  are  oval  and  usually  nearer  one  end  of  the  cell.  They 

1  Rosenau  and  Anderson:  U.  S.  Hygienic  Laboratory,  Bulletin  No.  43,  1908,  p.  59. 
The  official  test  dose  of  toxin  is  100  times  the  amount  of  a  dry  tetanus  toxin  required 
to  kill  a  350  gram  guinea-pig  in  four  days. 

2  Editorial,  Jour.  A.  M.  A.,  1909,  Vol.  LIU,  p.  955. 


284  SPECIFIC  MICRO-ORGANISMS 

are  only  feebly  resistant,  being  killed  at  85°C.  in  15  minutes  and 
by  5  per  cent  carbolic  acid  in  24  hours. 

Strict  anaerobiosis  is  necessary  for  successful  culture,  except 
when  B.  botulinus  grows  in  symbiosis  with  aerobes.  Growth  is 
best  at  25-30°  C.,  very  slight  at  37°-38.5°  C.,  and  best  in  a 
medium  slightly  alkaline  to  litmus.  Gelatin  is  quickly  liquefied 
and  abundant  gas  is  produced  in  glucose  media.  The  organism 
appears  to  be  incapable  of  growth  in  the  animal  body.  Cultures 
are  very  poisonous  when  injected  into  or  fed  to  animals. 

The  poison  "Botulin"  resembles  in  some  of  its  properties  the 
tetanus  toxin.  It  is  destroyed  rapidly  at  yo0-8o°  C.,  and  pre- 
serves its  toxicity  for  years  when  dried.  It  is  neutralized  by 
mixing  with  brain  substance.  It  differs  from  the  other  pow- 
erful toxins,  however,  in  its  ability  to  resist  the  gastric  juice  and 
to  poison  by  absorption  through  the  alimentary  canal.  Forssman 
has  immunized  guinea-pigs,  rabbits  and  goats,  and  has  obtained 
an  antitoxic  serum  from  these  animals. 

Botulism  is  a  form  of  food  poisoning  definitely  recognized  as 
such  as  early  as  1820  It  has  followed  the  consumption  of  sau- 
sage, hams,  fish  and  other  cured  or  preserved  meats.  The  symp- 
toms are  very  characteristic,  appearing  in  18  to  48  hours  after 
ingestion  of  the  poisonous  food.  There  is  vomiting,  dryness  of 
the  mouth  and  constipation,  motor  paralysis,  especially  early  in 
the  external  ocular  muscles.  The  involvement  of  the  central 
nervous  system  may  progress  to  complete  motor  paralysis  and 
death.  The  mind  is  usually  clear  even  in  the  fatal  cases.  This 
disease  is  evidently  due  to  the  poisons  already  formed  in  the  food 
at  the  time  it  is  eaten,  and  it  is  to  be  regarded  as  an  intoxication 
rather  than  an  infection.  Van  Ermengem  designates  B.  botu- 
linus as  a  pathogenic  saprophyte. 


CHAPTER  XIX. 

BACTERIACE^:  THE  BACILLUS  OF  DIPHTHERIA  AND 
OTHER  SPECIFIC  BACILLI  PARASITIC  ON  SUPER- 
FICIAL MUCOUS  MEMBRANES. 

Bacillus  (Bacterium)  Diphtherias. — Klebs  in  1883  discovered 
this  organism  in  the  microscopic  study  of  pseudomembranes 
from  fatal  cases  of  epidemic  diphtheria.  Loffler  in  1884  obtained 
pure  cultures  of  the  bacillus  and  by  inoculating  the  abraded 
mucous  membrane  of  susceptible  animals  with  his  cultures,  he 
produced  local  lesions  similar  to  those  observed  in  human  diph- 
theria, in  some  instances  followed  by  death  or  paralysis. 

B.  diphtheria  occurs  in  the  exudate  (false  membrane)  which 
occurs  in  the  pharynx,  larynx  and  adjacent  mucous  membranes 
in  epidemic  diphtheria,  on  the  mucous  membranes  of  those  who 
have  recovered  from  the  disease  and,  much  less  commonly,  on 
the  mucous  membranes  of  healthy  throats.  It  is  a  rod-shaped 
organism  extremely  variable  in  size,  shape  and  staining  properties. 
The  width  is  ordinarily  between  0.3  and  o.8/*  and  the  length 
varies  from  i  to  6ju.  The  cell  is  straight  or  slightly  curved  and 
very  frequently  of  uneven  diameter,  with  swelling  at  one  end  or 
in  the  middle  portion.  The  cell  contents  stains  unevenly  in 
many  of  the  cells.  Many  different  morphological  types  are  thus 
presented  which  may  be  designated  roughly  as  regular  cylinders, 
clubs,  spindles  and  wedges  according  to  form,  and  as  uniformly 
pale,  uniformly  dark,  regularly  or  irregularly  banded  or  granular 
according  to  internal  structure  of  the  stained  cell.  These  varia- 
tions in  form  and  internal  structure  are  best  seen  after  staining 
the  bacillus  with  Loffler's  methylene  blue  and  are  especially 
valuable  in  the  quick  recognition  of  B.  diphtheria  as  it  grows  in 
the  diphtheritic  membrane  or  in  culture  on  Loffler's  blood  serum. 

285 


286 


SPECIFIC  MICRO-ORGANISMS 


On  other  media,  such  as  glycerin  agar,  the  morphological  irregulari- 
ties are  less  marked  as  a  rule.     The  organism  in  young  cultures 


FIG.  116. — Bacillus  of  diphtheria.     X  1000. 


/ 


FIG.  117. — B.  diphtheria  stained  by  Neisser's  method. 

stains  readily,  best  perhaps  with  Loffler's  methylene  blue  in 
the  cold.  It  is  Gram-positive.  Old  cultures  stain  with  great 
difficulty. 


BACTERIACE.E :    THE   BACILLUS    OF   DIPHTHERIA 


287 


Loffler's  blood  serum  is  the  medium  of  choice.  The  colonies 
develop  at  37°  C.  in  8  to  12  hours  as  grayish,  slightly  elevated 
points  and  become  2  to  3  mm.  in  diameter  in  the  course  of  48 

A  B 


t>x 


FIG.  118. — Forms  of  B.  diphtheria  in  cultures  on  Loffler's  serum.  A,  Charac- 
teristic clubbed  and  irregular  shapes  with  irregular  staining  of  the  cell  contents. 
Xnoo.  B,  Irregular  shapes  with  even  staining.  X  1000.  (After  Park  and 

Williams.) 

hours.  Contiguous  colonies  become  confluent.  On  glycerin 
agar  after  24  hours  at  37°  C.,  the  colony  is  coarsely  granular 
with  a  somewhat  jagged  outline.  Many  variations  from  this 


FIG.  119. — Forms  of  B.  diphtheria  in  cultures  on  agar.  A,  Bacilli^small  and 
uniform.  Xiooo.  B,  Spherical  forms  in  culture  24  hours  old.  On  Loffler's  serum 
this  same  organism  produced  granular  forms.  X 1410.  (After  Park  and  Williams.) 

typical  appearance  occur.  Growth  in  gelatin  is  slow  and  ceases 
below  20°  C.  The  medium  is  not  liquefied.  The  bacillus  grows 
in  milk  without  producing  coagulation.  In  broth  the  growth 


288  SPECIFIC   MICRO-ORGANISMS 

may  occur  as  a  granular  sediment,  as  a  diffuse  cloudiness  or  as  a 
pellicle  on  the  surface,  depending  upon  the  reaction  and  pepton 
content  of  the  medium  and  the  vigor  of  growth  of  the  culture. 
The  growth  on  the  surface  produces  the  best  yield  of  toxin. 
Acid  is  produced  in  dextrose  broth.  The  organism  is  killed 
when  moist  by  heating  to  60°  C.  for  20  minutes.  It  is  fairly 
resistant  to  drying  and  has  been  found  alive  in  bits  of  dry  diph- 
theritic membrane  after  four  months. 

Roux  and  Yersin  in  1888  filtered  broth  cultures  of  the  diph- 
theria bacillus   through  porcelain  filters  and  found  the  filtrate 


FIG.  120. — Colonies  of  B.  diphtheria  on  agar.     X2oo.     (After  Park  and  Williams.) 

extremely  poisonous.  By  injecting  it  into  animals  they  were 
able  to  produce  the  signs  of  local  and  general  intoxication  which 
are  observed  in  the  natural  disease.  A  favorable  medium  for 
toxin  production  is  a  veal  broth  containing  2  per  cent  pepton 
and  having  a  titre  of  9  c.c.1  of  normal  sodium  hydroxide  above 
the  neutral  point  to  litmus.  It  should  be  placed  in  flasks  in  a 
thin  layer  to  allow  abundant  air  supply.  Incubation  for  from 
5  to  10  days  gives  the  maximum  toxicity.  The  filtrate  from  such 
a  culture  may  kill  a  250  gram  guinea-pig  in  a  dose  of  0.002  c.c. 
Less  powerful  toxin  is  frequently  obtained,  so  that  sometimes 
even  0.5  c.c.  or  more  may  be  required  to  kill  a  guinea-pig,  and 

1  Per  1000  c.c.  of  the  medium. 


BACTERIACE^E :    THE   BACILLUS    OF    DIPHTHERIA 


289 


some  strains  of  bacilli  morphologically  indistinguishable  from 
B.  diphtheria  seem  to  produce  no  toxin  at  all.  The  toxin  is 
quickly  destroyed  by  boiling  and  loses  95  per  cent  of  its  strength 
in  five  minutes  at  75°  C.  It  grad- 
ually deteriorates  even  at  low  tem- 
peratures. Its  chemical  nature  is 
unknown.  Ehrlich  has  shown  that 
old  toxin  which  has  lost  much  of  its 
poisonous  property  is  still  able  to 
combine  with  as  much  antitoxin  as 
before.  This  deteriorated  toxin  is 
called  toxoid.  He  explains  the  phe- 
nomenon by  assuming  the  existence 
of  two  distinct  chemical  groups  in 
the  toxin  molecule,  one  serving  to 
combine  with  antitoxin  and  being 
relatively  stable,  the  other  bearing 
the  poisonous  properties  and  readily 
undergoing  disintegration.  The 
former  he  has  called  the  haptopho- 
rous  group  and  the  latter  the  toxo- 
phorous  group.  In  toxoid  the  toxo- 
phorous  group  has  degenerated. 

Diphtheria  was  recognized  as  a 
distinct  disease  by  Bretonneau  in 
1821.  It  is  characterized  by  a  local 
inflammation,  usually  on  the  mu- 
cous membrane  of  the  throat,  the 
nose,  more  rarely  the  genital  mu- 
cous membrane,  or  the  surface  of 
a  wound,  and  by  an  accompanying  general  intoxication  giving  rise 
to  focal  necrosis  in  various  parenchymatous  organs  and  affecting 
more  particularly  the  heart  and  the  nervous  system.  The  local 
inflammation  may  be  only  a  mild  reddening  or  it  may  be  a  wide- 
spread area  of  necrosis.  Most  frequently  there  is  an  exudate 

I9 


FIG.  121. — B.  diphtheria,  culture 
on  glycerine  agar. 


2  go 


SPECIFIC   MICRO-ORGANISMS 


of  plasma  containing  leukocytes,  epithelial  cells  and  bacteria, 
and  this  coagulates  on  the  mucous  surface.  The  epithelium 
underneath  also  undergoes  necrosis  in  moderately  severe  cases 
and  is  firmly  attached  to  the  exudate  by  the  fibrin  threads.  In 
severer  forms  there  is  an  escape  of  blood  into  the  exudate  giving 
it  a  dark  color.  The  local  lesion  is  largely  due 
to  soluble  toxin  formed  by  the  bacilli.  The  gen- 
eral disturbance  is,  as  a  rule,  due  solely  to  the 
absorbed  toxin.  The  bacilli  remain  at  the  site  of 
the  lesion  and  do  not  appear  in  the  blood  or  in- 
ternal organs  in  any  appreciable  numbers.  They 
are  occasionally  found  in  the  spleen  or  kidney 
of  fatal  cases,  but  not  more  frequently  than  the 
streptococcus  is  found  in  these  organs  in  appar- 
ently uncomplicated  fatal  cases  of  diphtheria. 

The  local  lesion  in  the  throat  may  be   simu- 
lated very  closely  by  inflammation  due  to  strep- 
tococci, but  the  general  manifestations  are  not 
duplicated  in  such  conditions.     Mixed  infection 
—Swab    with  diphtheria  bacilli  and  virulent  streptococci 
and   culture-tube    mav  present  a  clinical  picture  of  great  severity. 

used  in  the  diag-  J-   J 

nosis  of  diphthe-  Bacteriological  examination  is  often  a  great  help 
oT'cotton  ondfhe  m  diagnosis  even  to  the  expert  clinician,  and  is 
wire  shown  is  quite  generally  employed. 

much  too  bulky.  „  .   7      .      7     ~  .  .          /•     r»  • ..  n  i      •          T 

Bacteriological  Diagnosis  of  Diphtheria. — In 
many  large  cities  the  bacteriological  diagnosis  of  diphtheria  is  un- 
dertaken by  boards  of  health.  The  methods  used  differ  somewhat 
in  detail,  but  are  similar  in  the  main,  and  are  based  upon  the  pro- 
cedure devised  by  Biggs  and  Park  for  the  Board  of  Health  of  New 
York  City.  Two  tubes  are  furnished  in  a  box.  The  tubes  are  like 
ordinary  test-tubes,  about  three  inches  in  length,  rather  heavy  and 
without  a  flange.  Both  are  plugged  with  cotton.  One  contains 
slanted  and  sterilized  Loffler's  blood-serum  mixture  (Fig.  122); 
the  other  contains  a  steel  rod,  around  the  lower  end  of  which  a 
pledget  of  absorbent  cotton  has  been  wound.  These  tubes  con- 


BACTERIACE.E :    THE   BACILLUS    OF   DIPHTHERIA  2QI 

taining  the  swabs  are  sterilized.  The  swab  is  wiped  over  the 
suspected  region  in  the  throat,  taking  care  that  it  touches  nothing 
else,  and  is  then  rubbed  over  the  surface  of  the  blood-serum  mix- 
ture. The  swab  is  returned  to  its  test-tube  and  the  cotton  plugs 
are  returned  to  their  respective  tubes.  The  plugs,  of  course, 
are  held  in  the  fingers  during  the  operation,  and  care  must  be 
taken  that  the  portion  of  the  plug  that  goes  into  the  tube  touches 
neither  the  finger  nor  any  other  object.  The  principles,  in  fact, 
are  the  same  as  those  laid  down  in  general  for  the  inoculation 
of  culture-tabes  with  bacteria  (see  page  107).  In  board-of-health 
work  these  tubes  are  returned  to  the  office.  When  it  is  desirable, 
a  second  tube  may  be  inoculated  from  the  swab.  The  tubes 
are  placed  in  the  incubator,  where  they  remain  for  from  6  to  15 
hours  and  a  microscopic  examination  is  then  made  of  smear 
preparations  stained  with  Loffler's  methylene  blue.  After  use 
the  tubes  and  swabs  should  be  most  carefully  and  thoroughly 
sterilized. 

On  Loffler's  blood-serum  kept  in  the  incubator  the  bacillus 
of  diphtheria  grows  more  rapidly  than  most  other  organisms 
which  are  ordinarily  encountered  in  the  throat,  a  property 
which  to  a  certain  extent  sifts  it  out,  as  it  were,  from  them,  and 
makes  its  recognition  with  the  microscope  easy  in  most  cases. 
The  appearance  of  the  bacilli  under  the  microscope  is  quite 
characteristic.  The  diagnosis  of  the  diphtheria  bacillus  in  prac- 
tice is  made  from  the  character  of  the  growth  upon  the  blood- 
serum  and  the  microscopical  examination,  taking  into  account 
the  size  and  shape  of  the  bacilli,  with  the  frequent  occurrence 
of  irregular  forms  and  the  peculiar  irregularities  in  staining,  and 
this  usually  suffices;  but  in  doubtful  cases  a  second  culture  should 
be  made  from  the  throat,  and  at  the  same  time  another  tube  of 
Loffler's  serum  should  be  inoculated  from  the  first  culture. 
On  the  next  day  plate  cultures  on  glycerin  agar  may  be  made 
from  which  typical  colonies  should  be  transplanted  to  broth. 
After  48  hours  at  37°  C.  the  broth  is  injected  into  two  guinea- 
pigs  in  doses  of  0.5  c.c.  and  one  of  the  guinea-pigs  should  receive 


2Q2  SPECIFIC   MICRO-ORGANISMS 

at  the  same   time  diphtheria  antitoxin.     In   this  way  virulent 
diphtheria  bacilli  may  be  accurately  detected. 

The  very  large  number  of  examinations  that  have  been  made 
by  various  boards  of  health  have  shown  that  the  diphtheria 
bacillus  may  persist  in  the  throat  for  a  long  time — occasionally 
several  weeks  after  the  patient  has  apparently  recovered;  also 
that  diphtheria  bacilli  are  occasionally  found  in  the  throat, 
when  there  is  an  inflammatory  condition  without  any  pseudo- 
membrane,  and  that  they  not  only  appear  in  an  apparently 
healthy  throat,  especially  in  hospital  nurses  and  in  children 
who  have  been  associated  with  cases  of  diphtheria,  but  also  in 
those  who  have  had  no  traceable  contact  with  diphtheria  cases.1 
It  has  been  found  that  bacilli  sometimes  occur  in  the  throat, 
which  have  all  the  morphological  and  cultural  properties  of  the 
diphtheria  bacillus,  but  which  are  devoid  of  virulence  when 
tested  upon  animals.  Such  diphtheria  bacilli  have  frequently 
been  called  pseudodiphtheria  bacilli.  A  bacillus  closely  resembling 
the  diphtheria  bacillus,  but  without  virulence,  has  been  found 
in  xerosis  of  the  conjunctiva.  It  is  called  the  xerosis  bacillus. 
If  not  a  transformed  diphtheria  bacillus,  it  is  at  least  closely 
related.  The  diphtheria  bacillus  is  subject  to  wide  variations 
in  morphology,  so  that,  in  dealing  with  unknown  cultures  where 
the  forms  are  not  characteristic  and  injection  into  animals  is 
without  result,  it  may  be  difficult  to  decide  whether  or  not  the 
organisms  are  diphtheria  bacilli. 

The  disease  is  undoubtedly  transmitted  very  largely  by 
immediate  contact,  especially  with  persons  harboring  the  bacilli 
but  not  seriously  ill,  and  by  fomites.  Children  in  school  or  at 
play  readily  transfer  secretions  of  the  mouth,  and  a  cough  or 
sneeze  may  distribute  such  material  over  a  wide  area. 
.  Immunity  to  diphtheria  was  produced  by  Von  Behring  in 
1890  by  injecting  the  toxin  into  animals,  the  general  method  of 
procedure  being  quite  similar  to  that  followed  in  the  production 
of  tetanus  antitoxin.  The  blood  serum  of  the  immunized  animal 

1  Sholly:  Journ.  Infect.  Dis.,  Vol.  IV,  1907,  pp.  337-346. 


BACTERIACE.E :    THE   BACILLUS    OF   DIPHTHERIA  293 

was  found  to  be  capable  of  neutralizing  the  poisonous  property 
of  diphtheria  toxin.  The  brilliant  success  of  Roux  (1884)  in  treat- 
ing diphtheria  with  antitoxic  serum  caused  the  rapid  adoption' 
of  antitoxin  as  a  therapeutic  agent  throughout  the  world.  Park 
and  his  co-workers,  Atkinson,  Gibson  and  Banzhaf,  have  devel- 
oped a  method  of  concentrating  diphtheria  antitoxin  which  is 
now  generally  employed. 

For  the  production  of  antitoxin1  young  healthy  horses  are 
selected  with  great  care.  They  are  specifically  tested  for  tubercu- 
losis and  glanders.  A  powerful  diphtheria  toxin  is  then  injected 
into  the  horses,  in  an  amount  sufficient  to  kill  5000  guinea-pigs, 
together  with  10,000  units  of  antitoxic  serum.  The  toxin  is 
subsequently  injected  at  intervals  of  three  days  and  each  succeeding 
dose  is  increased  by  about  20  per  cent  as  long  as  the  condition 
of  the  horse  is  satisfactory.  At  the  end  of  two  months  the  dose 
is  about  fifty  times  as  large  as  the  initial  dose.  Antitoxin  is 
given  only  at  the  start.  The  serum  of  the  horse  is  tested  from 
time  to  time  and,  when  the  desired  antitoxic  strength  has  devel- 
oped, the  blood  is  drawn  once  a  week  for  the  preparation  of  anti- 
toxin. A  dose  of  toxin  is  given  after  each  weekly  bleeding. 
The  blood  is  drawn  from  the  jugular  vein  into  jars  containing  a 
10  per  cent  solution  of  sodium  citrate,  nine  parts  of  blood  to  one 
of  the  citrate  solution.  The  material  is  mixed  and  allowed  to 
sediment  in  a  refrigerator.  The  plasma  is  then  siphoned  off 
into  large  bottles  and  heated  to  57°  C.  for  18  hours  to  change 
part  of  the  soluble  globulins2  to  euglobulins,  insoluble  in  a  satu- 
rated solution  of  sodium  chloride.  An  equal  volume  of  saturated 
aqueous  solution  of  ammonium  sulphate  is  then  added.  The 
precipitate  which  forms  consists  of  the  globulins  and  nucleopro- 
teins  of  the  plasma.  This  precipitate  is  collected  on  a  filter 
and  extracted  with  a  saturated  solution  of  sodium  chloride,  in 
which  the  pseudoglobulin  fraction,  carrying  with  it  the  antitoxic 

1  For  details  of  the  method  see  Park  and  Williams,  Pathogenic  Bacteria  and 
Protozoa,  Phila.,  1910. 

2  Banzhaf:  The  Preparation  of  Antitoxin;  Johns  Hopkins  Hosp.  Bull.,  1911, 
Vol.  XXII,  pp.  106-109. 


2Q4  SPECIFIC   MICRO-ORGANISMS 

property,  is  dissolved.  This  is  precipitated  by  the  addition  of 
dilute  acetic  acid,  filtered  out  and  again  taken  up  in  salt  solu- 
tion. It  is  carefully  neutralized  with  sodium  carbonate  and 
dialyzed  for  several  hours  against  water  to  remove  the  inorganic 
salts.  The  residue  in  the  dialyzer  is  then  passed  through  a 
Berkfeld  filter  to  sterilize  it,  a  preservative  is  added,  and  it  is 
ready  to  be  tested  and  put  up  in  containers  for  distribution. 
The  final  product  contains  75  to  90  per  cent  of  the  original  anti- 
toxic strength  and  is  only  about  one-third  as  bulky.  The  serum 
albumin,  euglobulin  and  nucleoprotein  have  also  been  to  a  large 
extent  eliminated  in  the  process  of  concentration. 

The  antitoxic  strength  of  anti-diphtheritic  serum  is  expressed 
in  immunity  units  and  is  ascertained  by  animal  experimentation. 
The  von  Behring  unit  is  contained  in  ten  times  the  amount  of 
serum  required  to  protect  a  250  gram  guinea-pig  perfectly  from 
the  effects  of  ten  times  the  dose  of  fresh  diphtheria  toxin  which 
kills  a  similar  guinea-pig  in  four  days.  The  dose  of  toxin  is 
first  ascertained  by  trial  on  guinea-pigs  and  the  dose  necessary 
to  kill  in  four  days  (minimum  lethal  dose)  determined.  Ten 
times  this  quantity  is  then  injected  along  with  varying  doses  of 
antitoxic  serum  into  a  series  of  guinea-pigs  until  the  quantity 
of  serum,  which  not  only  saves  the  animal  but  prevents  loss  of 
weight  and  local  induration  at  the  site  of  injection,  has  been 
ascertained.  Ten  times  this  amount  contains  one  immunity  unit. 

Ehrlich  has  carefully  standardized  an  antitoxic  serum  and 
has  preserved  it  as  a  dry  powder,  of  which  one  gram  contains 
1700  immunity  units.  This  standard  is  now  employed  as  the 
official  standard  for  comparison  in  the  United  States.  In  stand- 
ardizing an  antitoxin  by  the  Ehrlich  method,  one  unit  of  the 
standard  antitoxin  is  injected  along  with  various  quantities  of  a 
toxin  to  ascertain  how  much  of  the  latter  is  required  so  that  the 
animal  dies  after  four  days.  This  dose  of  toxin,  which  when 
combined  with  one  unit  of  the  standard  antitoxin,  kills  a  250 
gram  guinea-pig  in  four  days  is  called  the  L+  dose.  One  next 
injects  this  L+  dose  along  with  varying  quantities  of  the  new 


BACTERIACE.E :    THE   BACILLUS    OF   DIPHTHERIA  2Q5 

antitoxin,  and  the  amount  of  the  latter  which  keeps  the  guinea- 
pig  alive  for  just  four  days,  or,  in  other  words,  produces  the  same 
effect  as  the  standard  unit,  is  known  to  contain  one  immunity 
unit.  In  the  United  States,  the  Hygienic  Laboratory  at  Washing- 
ton furnishes  standard  antitoxin  to  manufacturers  for  this  official 
test  and  all  marketed  sera  are  tested  by  this  method. 

Diphtheria  antitoxin  not  only  prevents  the  development  of 
diphtheria  when  injected  in  doses  of  1000  units,  but  it  also 
exerts  a  marked  influence  as  a  therapeutic  agent  in  diphtheria, 
neutralizing  the  poison  produced  by  the  bacilli  in  the  body  of 
the  patient.  It  does  not  kill  the  bacilli  but  it  nullifies  their 
chief  offensive  weapon,  the  soluble  diphtheria  toxin.  Its  value 
in  treatment  of  diphtheria  is  everywhere  attested  by  clinical 
evidence.  The  inflammation  in  the  throat  subsides  and  the 
membrane  disappears.  The  bacilli,  however,  may  remain  for  a 
considerable  time.  Local  antiseptics  may  assist  the  natural 
agencies  of  the  body  in  their  destruction.  In  some  cases  they 
persist  for  months  in  spite  of  vigorous  treatment. 

Certain  untoward  effects  have  followed  the  injection  of  anti- 
diphtheritic  serum.  Sudden  death  has  occurred  in  very  rare 
instances  and  skin  rashes  are  rather  common.  These  effects 
are  probably  due  to  toxic  substances  set  free  in  the  parenteral 
digestion  of  the  foreign  protein  and  are  doubtless  of  the  same 
general  nature  as  the  phenomenon  of  anaphylaxis.  Since  the 
introduction  of  the  concentrated  antitoxin  fatalities  have  become 
exceedingly  rare  or  have  been  entirely  eliminated.  The  serum 
rashes  and  cases  of  nervous  shock  do  occur,  especially  in  asthmatic 
individuals  and  in  those  who  have  received  a  previous  injection 
of  horse  serum.  In  these  persons  it  is  well  to  give  a  minute 
quantity,  0.2  c.c.,  of  the  serum  as  a  preliminary  injection,  wait 
two  or  three  hours  and  then  give  the  full  dose.  The  danger  of 
serious  reactions  due  to  anaphylaxis  may  thus  be  avoided.1 

Bacillus  (Bacterium)  Xerosis. — This  organism  occurs  on  the 
normal  mucous  membranes,  particularly  the  conjunctiva.  It 

1  Vaughan:  Amer.  Journ.  Med.  Sciences,  1913,  Vol.  CXLV,  pp.  161-177. 


296  SPECIFIC  MICRO-ORGANISMS 

resembles  B.  diphtheria  very  closely,  simulating  the  granular 
morphological  type.  Its  cultures  are  not  poisonous. 

Bacillus  Hofmanni. — This  organism  is  also  called  the  pseudo- 
diphtheria  bacillus.  It  occurs  frequently  in  cultures  from  the 
nose  and  pharynx,  and  resembles  the  short  solid-staining  morpho- 
logical types  of  B.  diphtheria.  It  does  not  produce  toxin,  nor 
does  it  produce  acid  from  dextrose. 

The  Morax-Axenfeld  Bacillus.— This  is  a  small  non-motile 
diplo-bacillus,  the  individuals  measuring  about  1X2^,  which 


FIG.  123. — The  Morax-Axenfeld  bacillus  in  the  exudate  of  conjunctivitis.     (From 
McFarland  after  Rymowitsch  and  Matschinsky.} 

occurs  in  one  form  of  epidemic  conjunctivitis.  It  can  be  cultured 
on  Loffler's  serum  which  it  liquefies,  and  the  disease  has  been 
produced  in  man  by  inoculation  with  pure  cultures. 

The  Koch-Weeks  Bacillus. — This  a  non-motile  rod  0.25^ 
wide  and  i  to  2/z  long,  which  occurs  in  epidemic  conjunctivitis. 
It  is  cultivated  with  difficulty  and  abundant  moisture  is  essential 
to  success.  Inoculation  with  pure  cultures  causes  conjunctivitis. 

Bacillus  (Bacterium)  Pertussis  (Bordet-Gengou  Bacillus).— 
Bordet  and  Gengou  in  1906  described  a  minute,  non-motile 
bacillus  0.3X1.5^  which  occurs  in  the  sputum  and  on  the  mucous 
membrane  of  the  trachea  and  bronchi  in  whooping  cough.  They 


BACTERIACE.E :    THE   BACILLUS    OF   DIPHTHERIA  297 

obtained  cultures  of  the  organism  on  blood  agar  and,  employing 
these  cultures  as  an  antigen,  they  demonstrated  an  antibody 
in  the  blood  of  patients  by  means  of  the  complement-fixation 
test.  Klimenko1  has  further  succeeded  in  producing  a  chronic 
catarrh  of  the  respiratory  passages  in  monkeys  and  puppies  by 
applying  pure  cultures  to  the  tracheal  mucosa.  The  bacillus 
is  a  minute  rod,  motionless,  stained  with  moderate  difficulty, 
and  Gram-negative.  It  occurs  in  large  numbers  between  the 
cilia  of  the  epithelial  cells  lining  the  trachea  and  bronchi  in  cases 
of  whooping  cough  where  it  mechanically2  interferes  with  the 


FIG.     124. — Koch-Weeks    bacillus    in    muco-pus    from    conjunctivitis.     X    1000. 
(From  Park  and  Williams  after  Weeks.} 

action  of  the  cilia  and  gives  rise  to  irritation.  It  is  an  obligate 
aerobe  and  at  first  grows  well  only  on  media  containing  blood, 
ascitic  fluid  or  other  protein.  Later  it  adapts  itself  to  artificial 
culture  on  ordinary  media.  Gelatin  is  not  liquefied. 

Bacillus  (Bacterium)  Influenzae. — Pfeiffer  in  1892  isolated  a 
small  bacillus  0.25^  wide  by  0.5  to  2.0^1  long  from  the  bronchial 
secretion  in  cases  of  epidemic  influenza.  The  bacillus  occurs  in 
enormous  numbers  in  acute  uncomplicated  cases  of  influenza 
in  the  nasal  and  bronchial  mucus.  It  is  non-motile,  aerobic, 

1  Centralbl.f.  Bakt.  Orig.,  1909,  Bd.  XLVIII,  S.  64-76. 

2Mallory:  Pertussis:  The  Histological  Lesion  in  the  Respiratory  Tract,  Journ. 
Med.  Rsch.,  1912,  Vol.  XXVII,  pp.  115-124;  Mallory,  Hornor  and  Henderson, 
Journ.  Med.  Rsch.,  1913,  Vol.  XXVII,  pp.  391-397. 


298  SPECIFIC   MICRO-ORGANISMS 

rather  difficult  to  stain  and  Gram-negative.  Cultures  are 
obtained  on  ordinary  agar  smeared  with  fresh  human  or  rabbit's 
blood  or  upon  a  mixture  of  blood  and  agar.  Hemoglobin  seems 
essential  to  growth.  The  bacillus  is  very  sensitive  to  drying, 
and  its  transmission  would  seem  to  occur  largely  through  close 
association,  and  the  scattering  of  moist  droplets  of  material 
from  the  nose  and  mouth  in  sneezing,  coughing  and  talking. 
The  cultures  are  toxic  for  rabbits  and  monkeys.  The  causal 
relation  of  B.  influenza  to  influenza  is  not  as  yet  fully  established. 
Conditions  resembling  influenza  very  closely  seem  to  be  caused 
by  other  organisms,  such  as  the  cocci. 

Bacillus  (Bacterium)  Chancri  (Bacillus  of  Ducrey).— Ducrey 
in  1889  found  a  short  bacillus  in  the  soft  venereal  sore  known 
as  chancroid,  obtained  it  in  pure  culture  and  produced  typical 
lesions  by  inoculation  in  man.  The  organism  is  about  0.5X1.5^, 
often  growing  in  threads.  It  grows  on  a  blood-agar  mixture  at 
37°  C.  Material  for  culture  should  be  obtained  from  an  un- 
broken pustule  or  from  a  chancroidal  bubo,  so  as  to  avoid  con- 
taminating organisms.  The  bacillus  possesses  very  little  resist- 
ance to  drying  or  to  germicides.  Successful  inoculation  experi- 
ments have  been  carried  out  on  man,  on  monkeys  and  on  cats. 
Other  organisms1  appear  to  produce  soft  chancre  in  the  absence 
of  the  Ducrey  bacillus  in  some  cases. 

1  Herbst  and  Gatewood:  Journ.  A.  M.  A.t  1912,  Vol.  LVIII,  pp.  189-191. 


CHAPTER  XX. 

BACTERIACE^E:    THE    TUBERCLE    BACILLUS    AND 
OTHER  ACID-PROOF  BACTERIA. 

Bacillus  (Bacterium)  Tuberculosis. — Robert  Koch  in  1882 
discovered  the  minute  rods  in  tuberculous  tissue,  planted  the 
tissue  on  slanted  inspissated  blood  serum  and  obtained  pure 
cultures  of  the  tubercle  bacillus,  inoculated  these  cultures  into 
animals  and  produced  typical  tuberculosis.  He  succeeded  in 
doing  this  with  natural  tuberculosis  of  man  and  many  other 
mammals  and  also  with  the  tuberculosis  of  birds.  Silbey  in 
1889  observed  with  the  microscope  morphologically  similar 
bacilli  in  a  snake.  Rivolta  and  Mafucci  in  1889  pointed  out  the 
differences  between  the  tubercle  bacillus  of  birds  and  that  of 
mammals  and  their  work,  together  with  subsequent  confirmatory 
investigations,  has  established  a  distinct  avian  type  of  tubercle 
bacillus,  B.  tuberculosis  var.  gallinaceus.  In  1897  Bataillon, 
Dubard  and  Terre  found  acid-proof  bacilli  in  definite  histological 
tubercles  in  a  fish  (carp),  obtained  cultures  and  recognized  it  as 
distinct  from  the  mammalian  form,  and  it  was  subsequently 
designated  as  B.  tuberculosis  var.  piscium.  Theobald  Smith 
in  1898  published  the  results  of  a  careful  and  extensive  com- 
parative study  of  tubercle  bacilli  from  human  sputum  and 
tubercle  bacilli  from  tuberculous  tissue  of  the  bovine  pearl 
disease  (tuberculosis),  and  pointed  out  distinct  differences  in 
morphology,  cultural  characters  and  virulence  between  the 
organisms  derived  from  the  two  sources.  The  mammalian 
tubercle  bacilli  were  thus  divided  into  two  types,  and  subsequent 
investigation  has  fully  justified  the  recognition  of  B.  tuberculosis 

299 


300  SPECIFIC   MICRO-ORGANISMS 

var.  humanus  and  B.  tuberculosis  var.  bovinus.  Some,  or  perhaps 
all  four  of  these  types  may  be  eventually  recognized  as  distinct 
species.  At  present  the  designation  as  types  or  varieties  seems 
more  appropriate. 

Bacillus  Tuberculosis  var.  Humanus. — This  organism  occurs 


FIG.  125. — Bacillus  tuberculosis  in  the  sputum  of  a  consumptive;  stained  by  Ziehl 
method.     X  2100.     (After  Kossel.} 

in  the  infiltrated  lung  in  human  phthisis  and  also  in  the  great 
majority  of  the  other  tuberculous  lesions  in  man.  In  the  ex- 
ternal world  it  does  not  grow  naturally  and  passes  there  a  more 
or  less  temporary  existence  in  discharges  from  the  body,  of  which 
the  most  important  is  the  sputum.  The  cell  is  about  0.4;*  in 
width  and  quite  variable  in  length,  0.5  to  S.oju.  The  longer 


BACTERIACE^E :    THE    TUBERCLE   BACILLUS  301 

forms  are  often  somewhat  bent,  and  they  frequently  contain 
refractile  granules.  When  stained  these  forms  have  a  beaded 
or  banded  appearance.  Spores  have  not  been  observed.  Branch- 
ing forms  occur  sometimes  in  cultures,  suggesting  a  close  relation 
to  actinomyces  and  streptothrix.  There  is  a  considerable  amount 
of  a  waxy  substance  in  the  body  of  the  bacillus,  which  makes  it 
difficult  to  stain  and  also  difficult  to  decolorize  after  it  has  been 
stained.  Hot  carbol-fuchsin  is  generally  employed,  applying 
it  for  one  to  two  minutes.  The  preparation  is  then  washed  and 


»*    '  »   J 

I  x;^;  v    . 

\»'r9<    ^         I 


FIG.  126. — Bacillus  tuberculosis,  from  a  pure  culture.     X  1000. 

decolorized  in  dilute  mineral  acid  (2  to  20  per  cent)  and  in  alcohol. 
Tissue  elements  and  most  other  materials  may  be  completely 
bleached  by  this  treatment,  leaving  the  tubercle  bacilli  still 
colored.  B.  tuberculosis  is  Gram-positive. 

Cultures  are  most  readily  obtained  by  transferring  bits  of 
tuberculous  tissue,  free  from  other  micro-organisms,  to  moist 
slants  of  inspissated  blood  serum  or  Dorset's  egg  medium.  If 
the  available  material  is  already  contaminated,  the  extraneous 
organisms  may  usually  be  eliminated  by  inoculating  it  into 
guinea-pigs  and  making  the  cultures  from  the  tuberculous  guinea- 


302 


SPECIFIC  MICRO-ORGANISMS 


pig  tissue,  about  four  weeks  later.1  The  tubes  may  be  sealed 
with  rubber  caps  or  paraffin  and  incubated  at  37°  C.  Better 
results  are  obtained  by  leaving  the  tubes  unsealed  and  in- 
cubating at  37°  C.  in  an  atmosphere  saturated  with  moisture, 
as  the  bacillus  is  a  strict  aerobe,  but  this  requires  special  care 
and  is  not  absolutely  essential  to  success.  After  two  or  three 
weeks  a  dry,  white  growth  is  developed  which  may  later  become 
folded.  Transplants  from  the  primary  culture  to  glycerin  agar, 
glycerin  broth  or  glycerin  potato  are  usually  successful.  Old 


FIG.  127. — Tubercle   bacillus   showing   branching   andjinvolution   forms.     (After 

Migula.} 

cultures  on  potato  and  agar  often  become  yellowish  or  even 
pink  in  color. 

The  chemical  composition  of  tubercle  bacilli  has  been  ex- 
tensively studied.  The  moisture  content  varies  from  83  to  89 
per  cent.  The  ash  (inorganic  salts)  amounts  to  about  2.6  per 
cent  of  the  dry  substance,  and  about  half  of  this  is  phosphor  c 

1  It  is  possible  to  cultivate  tubercle  bacilli  directly  from  contaminated  material, 
such  as  sputum,  by  carefully  washing  it  in  sterile  water  and  then  spreading  it  over 
the  surfaces  of  a  series  of  serum  tubes.  Results  are  somewhat  uncertain.  For 
details  of  this  and  other  methods  see  Kolle  and  Wassermann,  Handbuch,  1912, 
Bd.  V,  S.  420-422. 


BACTERIACE.E :   THE   TUBERCLE  BACILLUS 


303 


acid  (PC>4).  The  waxy  constituent  of 
the  bacterial  cells  is  of  particular  in- 
terest. This  makes  up  from  8  to  40  per 
cent  of  the  dry  substance,  less  in  young 
and  more  in  old  cultures.  The  acid-proof 
staining  property  depends  upon  this 
waxy  substance,  for  the  bacilli  from 
which  it  has  been  extracted  by  ether-al- 
cohol are  no  longer  acid-proof  while  the 
wax  itself  exhibits  this  peculiarity  of 
staining.  It  is  also  known  that  the  ba- 
cilli in  young  cultures  are  on  the  whole 
less  acid-proof  than  those  from  old  cul- 
tures in  which  chemical  analysis  shows 
a  greater  concentration  of  the  waxy  sub- 
stance. The  protein  substances,  largely 
nuclein,  make  up  about  25  per  cent  of 
the  dry  cell  substance.  Several  other 
constituents  of  the  cell  have  been  iden- 
tified. As  in  the  case  of  other  bacteria 
the  chemical  composition  varies  within 
rather  wide  limits  according  to  the  nutri- 
tive medium,  conditions  of  growth  and 
especially  the  age  of  the  culture. 

The  poisons  of  the  tubercle  bacillus 
exist  to  a  large  extent  in  an  inactive 
form  in  the  culture  fluid  and  more 
particularly  as  an  undissolved  constituent 
of  the  bacterial  cell  bodies.  Culture  fil- 
trates exert  little  or  no  effect  upon  in- 
jection into  normal  animals.  The  dead 

FIG.  128— Bacillus  tuber- 

bacilli,  however,  give  rise  to  local  mflam-    culosis.    Culture  on  glycerin 
mation  and  in  many  instances  stimulate    agar    several    month 
the  formation  of  typical  tubercles  at  the 
point  where  they  lodge.     Evidently  the 


(From  McFarla  nd  after 
Curtis.) 


304  SPECIFIC   MICRO-ORGANISMS 

poison  is  set  free  from  some  substance  in  the  dead  cells  by  the 
action  of  the  tissue  cells  or  body  fluids  upon  them,  and  it  is  quite 
certain  that  the  bacteria-free  culture  fluid  (old  tuberculin)  becomes 
toxic  as  a  result  of  such  an  action. 

Tubercle  bacilli  outside  the  body  are  moderately  resistant 
to  harmful  influences.  In  dried  sputum,  they  have  been  found 
alive  after  eight  months.  Direct  sunlight  kills  the  bacilli  in 
sputum  in  a  few  minutes  if  this  be  exposed  in  a  thin  transparent 
layer.  In  thicker  masses  the  effect  of  light  is  uncertain.  In 
buried  cadavers  the  bacilli  remain  alive  and  virulent  for  2  to  6 
months.  In  watery  suspensions  the  bacilli  are  killed  by  heating 
to  60°  C.  for  15  minutes.  In  milk,  heating  at  60°  C.  for  20  minutes 
or  at  65°  C.  for  15  minutes  kills  the  tubercle  bacilli,  provided  all 
the  fluid  is  heated  to  this  temperature  for  the  full  period.  The 
bottle  should  be  tightly  stoppered  and  completely  immersed 
in  the  hot  water.  Dry  heat  at  100°  C.  for  30  minutes  is  effective. 
Against  chemical  disinfectants  B.  tuberculosis  is  rather  resistant, 
doubtless  because  of  the  waxy  constituent  of  the  cells.  Absolute 
alcohol  and  mercuric  chloride  i  to  500  fail  to  disinfect  sputum 
in  24  hours.  Five  per  cent  carbolic  acid  is  effective  in  this  time. 
Formalin,  5  per  cent  solution,  requires  about  12  hours.  B. 
tuberculosis  remains  alive  in  strong  antiformin  solutions  (a  pro- 
prietary preparation  of  chlorinated  caustic  alkali)  for  30  to  60 
minutes,  whereas  ordinary  bacteria  are  rapidly  disintegrated 
by  this  chemical  agent. 

Tuberculin  is  a  name  applied  to  various  chemical  products 
of  the  tubercle  bacillus.  The  oldest  and  most  important  tuber- 
culin was  described  by  Koch  in  1890.  It  is  made  by  growing 
the  bacillus  on  the  surface  of  4  per  cent  glycerin  broth  in  shallow 
flasks  at  37°  C.  for  eight  to  ten  weeks,  steaming  the  cultures 
for  one  hour  and  filtering  through  porcelain,  or  often  merely 
through  paper,  to  remove  the  dead  bacilli.  The  filtrate  is  then 
concentrated  to  one-tenth  its  original  volume  by  evaporation  at 
90°  on  the  water-bath.  The  product  keeps  indefinitely  in  sealed 
containers  and  is  known  as  Koch's  old  tuberculin  ("  alt  tuber- 


BACTERIACE.E :   THE    TUBERCLE  BACILLUS  305 

kulin").  Chemical  study  of  tuberculin  has  shown  that  the  spe- 
cific active  substance  is  a  thermostable,  dialyzable  substance, 
insoluble  in  alcohol,  which  gives  most  of  the  protein  reactions 
but  not  the  biuret  test.  It  is  digested  by  pepsin  and  by  trypsin.1 
Koch's  new  tuberculin,  better  known  as  tuberculin  B .  E.  ("Bacillen- 
emulsion")  is  made  from  the  solid  bacterial  growth  on  glycerin 
broth.  The  growth  is  pressed  between  filter  papers,  dried  and 
then  pulverized  in  a  ball  mill  for  about  three  months,  then  sus- 
pended in  50  per  cent  aqueous  solution  of  glycerin,  0.002  gram 
of  the  powder  to  each  cubic  centimeter.  Finally  it  should  be 
sterilized  by  heating  to  60°  C.  for  20  minutes.  This  tuberculin 
is  a  suspension,  not  a  solution,  and  must  be  thoroughly  mixed 
each  time  before  use.  Numerous  other  tuberculins  have  been 
prepared,  of  which  perhaps  the  " Bouillon  filtre"  of  Denys  is 
the  most  important.  It  is  the  porcelain  filtrate  of  the  unheated 
glycerin-broth  culture  of  the  tubercle  bacillus.  It  resembles 
Koch's  old  tuberculin  except  that  it  is  not  heated  and  is  not 
concentrated. 

Inoculation  of  animals  with  B.  tuberculosis  gives  rise  to  typical 
tuberculous  lesions  and  death  in  most  mammalian  species. 
The  guinea-pig  is  very  susceptible  to  subcutaneous  injection 
but  not  readily  infected  by  the  alimentary  route.  The  lesions 
are  usually  well  developed  four  or  five  weeks  after  subcutaneous 
inoculation  and  death  occurs  as  a  rule  in  6  to  12  weeks.  Rabbits 
are  less  susceptible  to  inoculation  with  the  human  type  and  they 
usually  recover  when  injected  with  small  doses  of  a  culture, 
o.oo i  gram  intravenously.  Cattle  are  quite  immune  to  this 
organism.  Large  doses  of  cultures  or  of  sputum  have  been 
injected  into  calves  and  older  bovines  without  producing  tubercu- 
losis, and  quarts  of  tuberculous  sputum  have  been  fed  to  bovine 
animals  with  negative  results. 

Tuberculosis  is,  economically,  the  most  important  human 
disease.  Approximately  one  death  in  every  three  between  the 
age  of  20  and  45,  the  active  period  of  life,  is  due  to  it.  It  was 
1  Lowenstein  in  Kolle  und  Wassermann,  Handbuch,  1912,  Bd.  V,  S.  554-  555. 

20 


306  SPECIFIC   MICRO-ORGANISMS 

recognized  as  a  contagious  disease  by  the  ancients.  Laennec,1 
in  1805,  by  extensive  post-mortem  studies  recognized  the  essential 
pathological  unity  of  tuberculous  processes.  Villemin,  in  1865, 
conclusively  demonstrated  its  transmissibility  by  successful 
inoculation  of  animals  with  tuberculous  tissue  from  man  and  from 
cattle. 

The  response  of  the  infected  tissue  to  the  presence  of  the 
tubercle  bacillus  results  in  a  localized  mass  of  granulation  tissue, 
the  tubercle,  of  which  the  histological  structure  is  so  characteristic 
that  the  presence  of  tuberculosis  may  be  recognized  by  it  alone. 
From  the  point  of  introduction  the  bacilli  may  be  distributed 
by  the  lymph  or  blood  stream  or  may  be  carried  by  wandering 
cells.  Eventually  a  bacillus  comes  to  rest  and  grows  slowly 
in  the  intercellular  spaces  of  connective  tissue.  Very  soon,  the 
neighboring  fixed  tissue  elements,  connective-tissue  cells  and 
endo  helial  cells,  begin  to  multiply  by  karyokinesis  and  at  the 
same  time  the  cells  become  swollen  with  nuclei  large  and  bladder- 
like,  forming  the  so-called  epithelioid  cells.  The  bacilli  are 
found  in  and  between  these  cells.  As  the  pathological  process 
continues  the  nucleus  of  an  occasional  epithelioid  cell  divides 
many  times  without  division  of  the  cytoplasm,  giving  rise  to  a 
multi-nucleated  giant  cell.  Very  early  in  its  development  the 
peripheral  portion  of  the  tubercle  becomes  infiltrated  with  lympho- 
cytes and  later,  as  the  giant  cells  are  formed,  numerous  poly- 
nuclear  leukocytes  are  also  present.  Newly  formed  blood  vessels 
are  absent.  With  further  extension,  the  center  of  the  tubercle 
undergoes  a  caseous  necrosis  and  liquefaction,  and  eventually 
this  necrotic  center  enlarges  so  as  to  break  through  an  epithelial 
surface  to  a  passage  to  the  exterior.  This  gives  rise  to  open 
tuberculosis  and  tubercle  bacilli  may  usually  be  found  in  the 
discharge  from  the  lesion  at  this  stage. 

The  tubercle  is  the  essential  histological  unit  of  tuberculosis. 
An  infiltrated  tissue  may  contain  myriads  of  these  tubercles  in 

1  For  a  history  of  tuberculosis  seeLandouzy:  Cent  ans  de  phtisiologie,  1808-1908, 
Sixth  Internat.  Cong,  on  Tuberculosis,  Special  Volume,  pp.  145-189. 


BACTERIACE.E :   THE   TUBERCLE  BACILLUS  307 

all  stages  of  evolution.  At  any  stage  in  its  evolution  the  develop- 
ment of  the  tubercle  may  become  arrested  and  it  may  retrogress- 
and  heal  if  the  infected  tissue  is  able  to  overcome  the  bacilli. 
If  this  occurs  early  the  bacilli  may  be  entirely  destroyed  and  the 
abnormal  tissue  may  disappear  completely  or  remain  only  as  a 
little  hyaline  or  fibrous  tissue.  After  caseation  has  occurred, 
healing  results  in  the  formation  of  a  dense  fibrous  nodule,  usually 
with  calcareous  material  in  the  center,  in  which  living  tubercle 
bacilli  can  usually  be  demonstrated. 

The  mode  of  infection  in  human  tuberculosis  has  been  a  matter 
of  some  controversy  and  much  of  the  evidence  concerning  it 
has  been  derived  from  animal  experimentation.  Unquestionably 
tubercle  bacilli  may  pass  through  epithelial  surfaces,  especially 
of  mucous  membranes,  without  production  of  any  demonstrable 
lesion.  Ingested  bacilli  readily  pass  through  the  intestinal 
mucosa,  especially  during  the  digestion  of  fat,  and  they  may 
first  produce  lesions  in  the  mesenteric  lymph  glands,  the  liver 
or  in  the  lungs.  In  the  latter  instance,  they  doubtless  pass  with 
the  absorbed  fat  through  the  thoracic  duct,  superior  vena  cava 
and  right  heart  to  the  pulmonary  arteries.  In  man,  the  most 
important  mode  of  infection  is  through  inhaling  the  dust  of  dry 
powdered  sputum,  as  a  result  of  which  lesions  develop  in  the 
lungs.  Tuberculosis  may  occur  in  any  tissue  of  the  body,  reach- 
ing it  through  the  blood  and  lymph.  A  massive  infection  of 
the  blood  stream  often  leads  to  generalized  miliary  tuberculosis 
with  minute  tubercles  in  all  the  organs. 

The  bacteriological  diagnosis  of  the  disease  depends  upon 
finding  the  tubercle  bacilli  in  discharges  from  the  suspected 
lesion.  In  sputum  an  acid-proof  bacillus  of  the  proper  size  and 
shape  is  almost  invariably  a  tubercle  bacillus  and  a  diagnosis 
based  upon  such  a  finding  by  an  experienced  microscopist  is 
justly  regarded  as  very  accurate.  Inoculation  of  guinea-pigs 
will  clinch  the  proof.  The  latter  procedure  will  also  sometimes 
detect  tubercle  bacilli  when  careful  microscopic  search  has  failed. 
In  discharges  from  the  intestine  or  urinary  organs  one  may 


308  SPECIFIC  MICRO-ORGANISMS 

meet  with  other  acid-proof  organisms  (B.  smegmatis),  and  more 
care  is  necessary  in  arriving  at  a  diagnosis.  In  tuberculous 
meningitis,  the  tubercle  bacillus  may  be  detected  by  microscopic 
examination  of  the  cerebrospinal  fluid1  in  nearly  every  case. 
The  filmy  clot  which  usually  forms  in  such  a  fluid  in  a  half  hour 
after  drawing  it  is  the  most  favorable  material  for  examination. 

When  a  considerable  amount  of  purulent  or  mucoid  material 
is  available  for  examination  and  one  has  failed  to  find  the  tubercle 
bacilli  by  the  usual  method  of  microscopic  examination,  it  is 
often  advisable  to  try  some  method  of  concentration.  One  of 
the  common  methods  of  general  applicability  is  that  of  Uhlenbuth, 
in  which  antiformin  is  employed  to  dissolve  the  tissue  elements, 
leaving  the  bacilli  unchanged.  LofHer's  modification2  of  the 
Uhlenbuth  method  is  a  convenient  one.  The  material  to  be 
examined  is  mixed  with  an  equal  amount  of  50  per  cent  anti- 
formin and  brought  to  a  boil.  This  dissolves  the  sputum  or 
other  material  and  serves  to  kill  the  bacilli.  It  is  then  cooled 
and,  for  each  10  c.c.,  1.5  c.c.  of  chloroform-alcohol  (i  19)  is  added. 
The  mixture  is  next  violently  shaken  to  form  a  fine  emulsion, 
and  is  then  centrifugalized  at  high  speed  for  15  minutes.  The 
solid  matter  collects  as  a  tough  mass  on  top  of  the  drop  of  chloro- 
form and  beneath  the  watery  liquid.  This  mass  is  crushed 
between  slides,  mixed  with  a  little  egg  albumen  or  with  some  of 
the  original  untreated  exudate,  spread,  fixed,  stained  and  exam- 
ined in  the  usual  way.  The  albuminous  material  is  necessary  to 
make  the  preparation  adhere  to  the  slide. 

Allergic  reactions  are  extensively  employed  in  the  diagnosis 
of  tuberculosis.  Tuberculin  is  without  particular  effect  upon 
normal  individuals  but  in  the  tuberculous  individual  it  gives 
rise  to  irritation  and  intoxication.  The  phenomenon  is  analogous 
to  that  of  anaphylaxis,  the  irritant  or  toxic  substance  being  set 
free  from  the  tuberculin  by  the  action  of  specific  ferments  pro- 


:  Amer.  Journ.  Dis.  Children,  Jan.  IQII,  Vol.  I,  pp.  26-36.  Hemenway: 
ibid.,  1911,  Vol.  I,  pp.  37-41.  Koplik:  Johns  Hopkins  Hosp.  Bull.,  1912,  Vol.  XXIII, 
pp.  113-120. 

2  Williamson:  Journ.  A.  M.  A.,  1912,  Vol.  LVIII,  pp.  1005-7. 


BACTERIACE^E :    THE    TUBERCLE   BACILLUS  309 

duced  and  present  in  the  body  as  a  result  of  previous  contact 
with  the  tubercle  bacillus  and  its  products.  The  tuberculous- 
individual  is  therefore  sensitized  to  tuberculin.  The  sensitization 
may  be  local  and  confined  to  the  tissue  immediately  surrounding 
a  solitary  tubercle,  or  it  may  be  general  as  a  result  of  more  ex- 
tensive lesions.  Tuberculin  is  applied  to  the  skin  mixed  with 
an  equal  amount  of  lanolin  (Moro  test),  or  applied  to  a  scarified 
point  undiluted  (Von  Pirquet  test),  or  injected  into  the  sub- 
stance of  the  skin  in  a  dose  of  o.i  c.c.  of  i  to  1000  dilution  (Ham- 
burger intracutaneous  test),  or  applied  to  the  conjunctiva  in  a 
dose  of  one  drop  of  a  freshly  prepared  i  per  cent  solution  of  old 
tuberculin  (Wolff-Eisner  or  Calmette  test),  or  finally  it  may  be 
introduced  into  the  circulation  by  subcutaneous  injection  of 
a  dilution  representing  o.ooooi  gram  of  old  tuberculin,  with 
subsequent  progressive  increase  of  the  dose  up  to  o.oio  gram  if 
reaction  is  not  obtained.  The  local  reaction  is  that  of  irritation, 
evidenced  by  redness  and  edema,  sometimes  by  vesiculation. 
The  general  reaction  is  evidenced  by  malaise,  irritation  at  site 
of  the  lesion  (increased  cough  in  pulmonary  tuberculosis)  and 
a  rise  in  body  temperature.  The  reaction  depends  upon  the 
tuberculin  coming  into  contact  with  the  specific  ferment,  and 
the  location,  extent  and  activity  of  the  tuberculous  process  are 
important  elements  influencing  the  outcome  of  the  various 
tests.  Tuberculosis  in  the  eye  causes  such  a  violent  reaction 
to  the  conjunctival  test  that  this  method  should  never  be  employed 
without  first  excluding  ocular  tuberculosis.  The  subcutaneous 
test  will  often  detect  tuberculosis  not  revealed  by  the  other 
methods.  It  is,  however,  a  more  serious  procedure  than  the  skin 
tests,  which  are  indeed  practically  harmless. 

The  various  tuberculins  are  now  extensively  employed  in 
the  treatment  of  tuberculosis,  largely  because  of  the  favorable 
results  obtained  by  Trudeau.  It  is  given  subcutaneously  every 
5  to  7  days  beginning  first  with  a  blank  dose  of  salt  solu- 
tion and  next  with  o.ooooi  gram  of  tuberculin.  The  dose  is 
kept  at  the  point  at  which  the  least  general  reaction  possibly 


310  SPECIFIC   MICRO-ORGANISMS 

recognizable  occurs,  or  just  below  this  amount,  the  general  pur- 
pose being  to  induce  an  immunity  to  tuberculin.  It  is  often 
possible  to  begin  with  a  case  which  reacts  to  o.oooi  gram  of  tuber- 
culin and  after  treatment  for  6  months  so  change  the  sensitive- 
ness that  0.5  gram  may  be  injected  without  reaction.  Some 
cases  do  remarkably  well  when  treated  with  tuberculin  together 
with  the  usual  careful  hygienic-dietetic  treatment1  given  in  sanito- 
ria,  but  the  value  of  tuberculin  for  treatment  of  the  average  case, 
is,  perhaps,  not  yet  fully  established.2 

Bacillus  Tuberculosis  var.  Bovinus. — The  bovine  type  of 
tubercle  bacillus  is  found  in  the  lesions  of  tuberculous  cattle 
(perlsuchi),  frequently  in  hogs,  in  a  considerable  percentage  of 
tuberculous  lesions  in  children,  and  very  rarely  in  the  tubercu- 
lous lungs  of  adult  human  beings.  In  artificial  culture  on  solid 
media,  the  cell  is  about  i/z  long,  shorter  than  that  of  the  human 
type,  and  is  easily  stained.  In  glycerin  broth  the  length  of  the 
cell  and  the  staining  is  more  irregular.  On  all  media  the  growth 
is  at  first  much  less  abundant  than  that  of  the  human  type. 
Smith  has  shown  that  the  bovine  type  produces  alkali  in  glycerin 
broth  during  the  first  two  months,  whereas  the  human  type 
tends  rather  to  .produce  acid.  The  virulence  of  the  bovine 
bacillus  is  greater  than  that  of  the  human  type  for  all  mammals, 
and  it  also  infects  birds.  Intravenous  injection  of  o.ooooi 
gram  of  culture  in  thin  emulsion  kills  rabbits  with  generalized 
tuberculosis  in  about  three  weeks,  while  a  similar  dose  of  the 
human  variety  is  without  such  effect.  Subcutaneous  injection  of 
rabbits  shows  a  similar  difference.  Calves  are  very  susceptible 
to  the  bovine  type,  not  to  the  human. 

Tuberculosis  of  cattle  is  widely  distributed  and  is  very  preva- 
lent in  the  older  European  dairy  regions.  The  lesions  are 
most  common  in  the  bronchial  and  retropharyngeal  lymph  glands, 
but  they  may  occur  anywhere  in  the  body  of  the  animal.  The 
disease  may  remain  localized  for  years  in  a  single  lymph  gland  or  it 

1  Brown:  Journ.  A.  M.  A.,  1912,  Vol.  LVIII,  pp.  1678-81. 

2  Brown:  Amer.  Journ.  Med.  Sciences,  1912,  Vol.  CXLIV,  pp.  469-624. 


BACTERIACE.E  I    THE    TUBERCLE   BACILLUS  31 1 

may  extend  rapidly  causing  marked  emaciation  and  death  of 
the  animal.  The  bacilli  escape  from  the  living  bovine  animal 
most  commonly  in  the  feces,1  sometimes  in  the  mucus  and  spray 
from  the  nose  and  mouth,  in  the  uterine  discharge  and  in  the 
milk,  and  of  great  importance  is  the  fact  that  animals  may  be 
excreting  the  bacilli  without  showing  any  gross  evidence  of  the 
presence  of  the  disease.  Tuberculin  is  extensively  employed  in 
the  detection  of  tuberculosis  in  cattle.  A  dose  of  0.2  to  0.5 
gram  diluted  with  9  volumes  of  0.5  per  cent  carbolic  acid  is  in- 
jected subcutaneously  at  the  side  of  the  neck.  The  typical 
positive  reaction  includes  a  rise  in  temperature  of  2°  or  3°  F. 
over  that  of  the  previous  day.  The  test  is  very  accurate  when 
positive  but  not  so  reliable  when  negative.  Tuberculous  animals 
should  be  segregated  from  healthy  animals  and  food  products 
from  them  used  only  after  effective  disinfection,  or  they  should 
be  slaughtered  under  inspection. 

Great  interest  has  been  manifested  in  the  question  of  suscep- 
tibility of  man  to  the  bovine  tubercle  bacilli  and  the  solution 
has  been  reached  by  isolating  bacilli  from  human  tissue  and  identi- 
fying them.  Park  and  Krumwiede2  have  summarized  the  results 
of  1511  such  examinations,  and  conclude  that  somewhat  less 
than  10  per  cent  of  the  deaths  from  tuberculosis  in  young  children 
are  due  to  the  bovine  tubercle  bacillus,  while  in  adults  infection 
with  this  bacillus  is  much  less  frequent. 

Bacillus  Tuberculosis  var.  Gallinaceus  (Avium). — This  variety 
occurs  particularly  in  the  tuberculous  lesions  of  barnyard  fowls, 
but  also  in  many  other  birds.  The  form  of  the  bacillus  is  not 
specially  characteristic  except  that  in  old  cultures  there  is  a 
marked  tendency  to  the  production  of  branching  threads.  In 
glycerin  broth  the  growth  is  more  delicate,  and  development 
takes  place  at  the  bottom  of  the  flask  as  well  as  on  the  surface 
of  the  liquid.  Chickens  are  very  susceptible  to  intravenous 
inoculation  with  this  type  of  bacilli  but  quite  refractory  to  the 

1  Briscoe  and  MacNeal:  111.  Agr.  Exp.  Sta.  Bull.  149,  1911;  Assn.  for  Tubercu- 
osis,  Transactions,  1912,  pp.  460-465. 

*Journ.  Med.  RscL,  1912,  Vol.  XXVII,  pp.  109-114. 


312  SPECIFIC   MICRO-ORGANISMS 

mammalian  types.  Mice  and  rabbits  are  also  susceptible,  while 
guinea-pigs  are  relatively  resistant.  The  avian  tubercle  bacillus  has 
been  found  in  human  tuberculous  lesions  in  a  very  few  instances. 

Bacillus  Tuberculosis  var.  Piscium.— This  variety  occurs 
in  natural  tuberculous  lesions  of  snakes,  fish,  turtles  and  frogs. 
The  bacillus  is  quite  different  from  the  preceding  varieties,  as 
it  grows  rapidly  on  ordinary  media  at  temperatures  ranging  from 
12°  to  36°  C.,  and  the  bacilli  developed  on  the  poorer  media  are 
often  not  at  all  acid-proof.  When  grown  in  bouillon  with  fre- 
quent shaking  the  culture  becomes  diffusely  cloudy,  and  the 
organisms  of  such  cultures  are  said  to  be  motile.  Most  warm- 
blooded animals  are  wholly  refractory  to  inoculation,  but,  in 
the  guinea-pig,  inoculation  has  sometimes  been  followed  by  the 
production  of  typical  tubercles  with  epithelioid  and  giant  cells, 
usually  encapsulated  and  tending  to  heal. 

Bacillus  (Bacterium)  Leprse. — Hansen  in  1873  and  Neisser 
in  1879  discovered  this  organism  in  the  nodular  lesions  of  leprosy. 
Cultures  were  first  obtained  by  Clegg  in  1908  by  inoculating 
leprous  tissue  onto  agar  along  with  living  amebae  and  the  vibrio 
of  Asiatic  cholera.  Pure  cultures  of  B.  leprcz  were  subsequently 
obtained  by  heating  the  mixture  to  kill  the  other  organisms. 
Inoculation  of  cultures  into  mice  and  guinea-pigs  is  said  to  pro- 
duce leprous  nodules  but  the  evidence  has  not  appeared  to  be 
very  convincing.  More  recently  Duval  and  Couret1  after  very 
extensive  investigations,  in  which  Clegg's  work  was  confirmed, 
have  been  able  to  produce  very  typical  leprosy  in  a  monkey  by 
repeated  injections  of  a  pure  culture,  resulting  in  general  dissemi- 
nation and  death  one  year  after  the  last  injection.  The  results 
have  not  been  confirmed  and  is  a  subsequent  paper2  Duval  is 
inclined  to  question  the  value  of  his  previous  animal  experiments, 
and  even  suggests  that  the  organism  employed  plays  only  a 
negligible  part  in  leprosy. 

B.  leprce  is  a  slender  rod  0.2  to  0.45/4  wide  by  1.5  to  6ju  long 

1  Journ.  Exp.  Med.,  Vol.  XV,  pp.  292-306. 

2  Duval  and  Wellman,  Journ.  Inf.  Diseases,  1912,  Vol.  XI,  pp.  116-139. 


B ACTERIACE^E :   THE    TUBERCLE  BACILLUS  313 

as  it  occurs  in  tissues,  much  shorter  in  cultures.  In  its  staining 
properties  it  closely  resembles  the  tubercle  bacillus,  but  is  less 
constantly  acid-proof  in  cultures.  The  organism  occurs  in 
enormous  numbers  in  most  of  the  nodular  lesions  of  leprosy  and 
if  often  abundant  in  the  nasal  mucus  of  these  cases.  When  less 
numerous  the  antiformin  method  of  Uhlenbuth  may  assist  in 
finding  them.  Duval  and  his  co-workers  have  obtained  pure 
cultures  by  the  method  of  Clegg  and  also  by  planting  uncontami- 
nated  leprous  tissue  on  serum  agar  to  which  trypsin  has  been 
added.  Eventually  the  bacilli  adapt  themselves  to  growth  on 
ordinary  media  such  as  plain  agar.  In  the  first  cultures  the  growth 
may  be  slow  and  relatively  meager,  but  later  abundant  growth 
may  be  obtained  in  2  to  3  days.  The  color  is  orange.  Injection 
of  these  cultures  into  mice,  guinea-pigs  and  monkeys  is  ordinarily 
followed  by  transient  lesions  which  have  been  considered  by  some 
to  resemble  those  of  leprosy.  The  one  instance  of  the  monkey 
reported  by  Duval  and  Couret,  mentioned  above,  seems  to  be 
more  convincing,  but  further  work  is  necessary  before  the  status 
of  these  cultures  can  be  definitely  established. 

Leprosy  has  been  known  since  the  dawn  of  history  and  has 
been  considered  to  be  transmissible  for  a  long  time.  It  is  widely 
distributed  over  the  earth,  especially  in  Norway,  Russia,  Iceland 
and  in  Turkey.  In  the  United  States  there  are  leper  colonies  in 
Louisiana,  Minnesota  and  in  Hawaii.  Lepers  are  occasionally  seen 
in  the  clinics  of  all  the  larger  cities. 

Leprosy  is  universally  considered  to  be  due  to  the  leprosy 
bacillus,  but  as  to  mode  of  transmission,  whether  direct  from 
man  to  man,  or  from  the  external  world,  or  how,  little  or  nothing 
is  really  known.  It  seems  certain  that  the  disease  is  always  con- 
tracted in  some  way  from  a  previous  case,  but  it  is  certainly  not 
very  readily  transmitted.  Segregation  without  absolute  isolation 
is  the  common  method  of  handling  lepers.  The  disease  is  not 
ordinarily  inherited. 

Bacillus  Smegmatis. — This  organism  occurs  in  the  smegma 
on  the  genitals  of  man  and  other  mammals  and  also  in  moist  folds 


314  SPECIFIC  MICRO-ORGANISMS 

of  the  skin  where  there  are  collections  of  moist  desquamated 
epithelium.  It  resembles  the  tubercle  bacillus  in  form  and  stain- 
ing properties,  but  is,  on  the  average,  more  readily  decolorized  in 
alcohol.  This  property  cannot  be  relied  upon  to  differentiate 
the  two  organisms  in  any  given  case.  Proper  care  in  collecting 
specimens  for  examination  usually  suffices  to  exclude  this  or- 
ganism. Urines  to  be  examined  for  tubercle  bacilli  should  be 
obtained  by  catheter.  In  doubtful  cases  inoculation  of  a  guinea- 
pig  is  necessary.  B.  smegmatis  has  been  grown  in  artificial  culture 
and  after  a  time  adapts  itself  to  ordinary  media. 

Bacillus  Moelleri. — Acid-proof  organisms  resembling  the 
tubercle  bacillus  in  form  and  staining  properties  were  found  on 
timothy  hay  by  Moeller.  The  bacillus  is  likely  to  be  found  in 
milk  and  other  dairy  products.  Probably  the  "  butter  bacillus" 
of  Rabinowitsch  is  identical  with  it  or  a  near  relative.  When 
introduced  into  guinea-pigs  these  organisms  sometimes  produce 
lesions  resembling  tubercles,  but  these  do  not  progress  and  kill 
the  animal  and  a  second  animal  inoculated  from  the  lesions  of  the 
first  gives  a  negative  result.  Cultures  are  easily,  obtained  on 
ordinary  media,  and  the  organisms  grow  rapidly  at  25°  to  30°  C. 

Other  Acid-proof  Organisms. — Many  of  the  strep  to  thrices 
which  grow  in  the  soil  and  upon  plants  are  to  some  extent  similar 
in  their  staining  properties  to  the  tubercle  bacillus  and  when 
broken  up  into  short  segments  may  be  a  source  of  confusion. 
These  are  most  likely  to  be  met  with  in  examining  agricultural 
products  and  especially  in  the  feces  of  cattle.  Mere  microscopic 
examination  of  such  materials  for  tubercle  bacilli  has,  as  a  rule, 
little  value,  as  both  positive  and  negative  findings  are  question- 
->able.  Brem,1  in  the  Canal  Zone,  has  made  the  important  obser- 
vation that  acid-proof  bacilli  may  grow  in  distilled  water  stored 
in  bottles  in  the  laboratory  and  that,  when  such  water  is  used  in 
preparing  the  microscopic  objects  for  examination,"  these  extrane- 
ous bacilli  may  be  mistaken  for  tubercle  bacilli.  Burvill-Holmes2 

1  Journ.  A.  M.  A.,  1909,  Vol.  LIU,  pp.  909-911. 

2Proc.  Path.  Soc.  Phila.,  1910,  N.  S.  Vol.  XIII,  pp.  154-160. 


BACTERIACE.E :    THE    TUBERCLE   BACILLUS  315 

has  made  similar  observations  at  Philadelphia.     Pseudo-bacilli, 
microscopic  bodies  somewhat  resembling  tubercle  bacilli,  some-- 
times  occur  in   microscopic  preparations   stained   with   carbol- 
fuchsin.     These  deceptive  pictures  seem  to  be  common  in  prepa- 
rations of  laked  or  digested  blood.1 

1  Calmette,  Sixth  Internat.  Cong,  on  Tuberculosis,  1908,  Spec.  Vol.,  p.  70;  see 
also  Bacmeister,  Kahn  and  Kessler,  Munch,  med.  Wochenschr.,  Feb.  18,  1913. 


CHAPTER  XXI. 

BACTERIACE^E:  THE  BACTERIA  OF  THE  HEMOR- 
RHAGIC  SEPTICEMIAS,  PLAGUE  AND  MALTA  FEVER. 

Bacillus  (Bacterium)  Avisepticus. — Moritz1  in  1869  observed 
this  minute  rod  in  the  blood  of  chickens  with  chicken  cholera. 
Toussaint  (1879)  and  Pasteur  (1880)  obtained  pure  cultures  in 
liquid  media  and  Pasteur  (1880)  made  the  far-reaching  discovery  of 
the  method  of  immunization  by  means  of  attenuated  bacterial 
cultures  while  working  with  this  organism.  B.  avisepticus  occurs  in 
enormous  numbers  in  the  blood,  internal  organs,  urine  and  feces  of 
the  acutely  affected  birds,  in  far  smaller  numbers  in  those  having 
the  chronic  form  of  the  disease  and  has  also  been  found  in  the  in- 
testinal contents  of  apparently  healthy  birds.  It  is  0.3^  wide  and 
0.2  to  iju  in  length,  the  shorter  ones  being  joined  together. 
It  is  non-motile  and  Gram-negative.  Cultures  are  readily  ob- 
tained on  ordinary  media  by  inoculation  with  heart's  blood. 
Gelatin  is  not  liquefied.  Minute  quantities  of  a  virulent  culture 
suffice  to  produce  a  fatal  infection  in  chickens  and  many  other 
birds,  either  by  feeding  or  by  subcutaneous  injection.  Rabbits 
are  also  extremely  susceptible,  guinea-pigs  almost  immune. 
Artificial  cultures  kept  for  three  to  ten  months  in  contact  with  air 
are  no  longer  capable  of  causing  a  fatal  infection  in  chickens  and 
their  injection  is  followed  by  recovery  and  a  state  of  immunity  to 
the  fully  virulent  organism.  Acute  chicken  cholera  is  the  typical 
hemorrhagic  septicemia  of  birds,  with  abundant  bacteria  in  the 
blood,  and  hemorrhages  on  the  serous  membranes  and  into  the 
stomach  and  intestine. 

Bacillus  (Bacterium)  Plurisepticus. — This  name  is  applied  to 
an  organism  occurring  in  the  hemorrhagic  septicemias  of  various 

1  Vallery-Radot:  Life  of  Pasteur,  1911,  Vol.  II,  p.  75. 

316 


THE   BACTERIA    OF    THE    HEMORRHAGIC    SEPTICEMIAS          317 

mammals  and  birds.  The  virulence  is  variable  and  seems  to  be 
especially  developed  for  the  species  of  animal  in  which  the  organ- 
ism is  found.  It  does  not  differ  essentially  from  B.  avisepticus. 
Other  minute  bacteria  exhibiting  the  same  general  characteristics 
and  occurring  as  a  generalized  infection  in  diseases  of  animals 
are  Bacillus  murisepticus  in  mice  and  Bacillus  (Bacterium)  rhusio- 
pathicz  suis  in  swine. 

Bacillus  (Bacterium)  Pestis. — This  organism  was  discovered 
simultaneously  by  Kitasato  and  Yersin  in  1894  in  the  bodies  of 
persons  dying  of  bubonic  plague  in  the  epidemic  at  Hongkong. 


FIG.  129. — Bacillus  of  bubonic  plague.     (Yersin.} 

The  description  of  Yersin  has  proven  to  be  the  more  accurate. 
The  organism  is  unquestionably  the  cause  of  plague,  as  in  addi- 
tion to  the  evidence  of  animal  experimentation  there  are  several 
instances  of  fatal  infection  of  men  working  with  the  organism 
in  laboratories  far  removed  from  any  focus  of  the  disease,  and 
finally  the  very  unfortunate  accident  at  Manila1  where  cholera 
vaccine  mixed  with  a  culture  of  B.  peslis  by  mistake  was  injected 
into  men  and  caused  fatal  bubonic  plague. 

B.  pestis  in  the  body  of  the  patient  is  a  short  plump  rod,  0.5 
to  0.7;*  wide  by  1.5  to  i.8/z  long,  and  rounded  at  the  ends.     When 

1  Freer:  Journ.  A.  M.  A.,  1907,  Vol.  XL VIII,  pp.  1264-65. 


318  SPECIFIC  MICRO-ORGANISMS 

stained  the  ends  become  deeply  colored  while  the  equator  remains 
pale  (bipolar  staining) .  Alongside  this  typical  form  many  irregu- 
lar organisms  are  usually  found,  especially  longer  and  shorter 
bacilli,  some  pale,  some  irregularly  outlined,  and  some  swollen 
and  poorly  stained.  The  last-mentioned  types  of  bacilli  are  more 
frequently  found  in  the  bodies  of  plague  victims  which  have  be- 
gun to  decompose.  They  are  also  observed  in  artificial  cultures. 
These  irregular  forms  (involution  forms)  are  important  in  the 
quick  recognition  of  plague.  The  bacillus  stains  very  readily, 
best  with  methylene  blue  or  with  a  momentary  exposure  to  carbol- 
fuchsin.  Better  results  are  obtained  by  fixing  the  spread  in  alco- 
hol one  minute,  rather  than  heating  it.  The  Romanowsky  stain 
gives  good  results.  It  is  distinctly  Gram-negative  (contrary  to 
the  original  statement  of  Kitasato).  Capsules  may  be  demon- 
strated on  bacilli  in  the  peritoneal  exudate  of  guinea-pigs  and 
mice,  less  easily  in  cultures.  It  is  non-motile  and  flagella  have 
not  been  demonstrated.  Spores  have  not  been  observed  and 
cultures  are  killed  at  60°  C.  in  10  to  40  minutes.  It  is  also  easily 
destroyed  by  chemical  germicides,  for  example,  by  5  per  cent  car- 
bolic acid  in  i  minute.  Mere  drying  at  35°  to  37°  C.  kills  the 
bacillus  in  two  to  three  days,  but  at  20°  C.  it  may  withstand  drying 
for  20  days.  It  may  live  for  months  in  frozen  material. 

Cultures  are  readily  obtained  on  ordinary  media,  best  at  a 
temperature  between  25°  and  30°  C.  Growth  is  moderately 
slow.  Gelatin  is  not  liquefied.  On  agar  containing  3  per  cent 
of  sodium  chloride,  irregular  involution  forms  areprodu  ced  in  24 
to  48  hours.  Long  chains  are  produced  in  broth.  It  does  not 
form  gas  from  sugars  but  does  produce  acid  from  dextrose,  levu- 
lose,  mannite  and  galactose,  not  from  lactose  or  dulcite. 

The  toxins  of  the  plague  bacillus  are  in  part  soluble  and  in 
part  intimately  combined  with  the  bacterial  cell.  Filtrates  of 
young  broth  cultures  are  without  toxic  properties  but  older  broth 
cultures  (14  days)  yield  a  toxic  filtrate.  The  bacterial  cells  killed 
by  heat  produce  fatal  poisoning  in  guinea-pigs  and  rabbits. 
The  poisons  obtained  so  far  are  much  less  powerful  than  the  sol- 


THE   BACTERIA   OF    THE    HEMORRHAGIC    SEPTICEMIAS          319 

uble  toxin  of  B.  diphtherias  or  the  endotoxins  of  the  typhoid  and 
cholera  germs. 

Rodents,  especially  rats  and  guinea-pigs,  are  very  susceptible 
to  inoculation,  even  a  needle  prick  carrying  the  minutest  quantity 
of  a  virulent  culture  being  sufficient  to  kill  in  a  few  days.  At 
autopsy  the  adjacent  lymph  nodes  are  found  greatly  swollen 
and  surrounded  by  hemorrhagic  edema.  The  spleen  is  greatly 
swollen.  Everywhere  are  enormous  numbers  of  the  bacilli. 
Infection  by  feeding  gives  positive  results  in  about  half  the  ex- 
periments. Inhalation  of  the  bacilli  produces  typical  pneumonic 
plague  in  rats.  Monkeys  are  susceptible  and  present  lesions 
similar  to  human  plague. 

Bubonic  plague  can  be  recognized  in  descriptions  of  epidemics 
in  very  ancient  records.  Rufus  of  Ephesus  who  lived  at  the  time 
of  Trajan  (A.  D.  98)  mentions  specifically  a  very  fatal  acute 
bubonic  plague  ("  pestilentes  bubones").  Great  epidemics  oc- 
curred in  Europe  in  the  6th  century  (527-565  A.  D.),  in  the  four- 
teenth century  (1347-1350  A.  D.).  Each  of  these  was  followed 
by  smaller  outbreaks  persisting  in  the  latter  epidemic  up  to  about 
1850.  It  is  estimated  that  25  million  persons  died  of  the  plague 
in  the  "  Great  Mortality"  of  the  i5th  century.  Another  pandemic 
of  plague  began  in  1893.  Its  progress  has  been  slow  and  un- 
doubtedly hampered  by  the  prophylactic  measures  made  possible 
by  the  discovery  of  Yersin  and  Kitasato.  It  exists  as  a  persistent 
infection  among  rodents  or  human  beings,  or  both,  in  central 
Asia,  central  China,  northern  India,  Arabia,  southern  Egypt, 
and,  more  recently,  seems  to  be  establishing  itself  in  California. 
Outbreaks  of  plague  in  man  in  new  localities  have  usually  been 
preceded  or  associated  with  mortality  among  rodents,  especially 
rats.  When  an  epidemic  begins  in  a  seaport  town,  the  sewer  rats 
(Mus  decumanus)  are  first  attacked.  Two  to  three  weeks  later 
the  house  rats  (Mus  rattus)  begin  to  die,  and  about  four  weeks 
later  the  epidemic  of  human  plague  begins.  The  transmission 
from  animal  to  animal  and  from  animal  to  man  is  accomplished 
very  largely  by  the  agency  of  fleas.  Rat  fleas  are  rarely  found 


320  SPECIFIC   MICRO-ORGANISMS 

on  man  or  at  large  in  human  habitations  as  long  as  their  normal 
hosts  are  at  hand,  but  when  the  rats  sicken  and  die  of  plague, 
then  the  fleas  leave  and  becoming  hungry  they  bite  human  beings 
and  thus  'noculate  them  with  plague  bacilli. 

In  its  permanent  endemic  centers,  plague  exists  as  an  acute 
and  chronic  disease  of  rodents.  It  spreads  from  these  regions 
through  the  agency  of  the  wandering  rats  traveling  along  the 
routes  of  commerce  and  especially  in  ships.  The  infected  rat, 
arrived  at  its  destination,  sets  up  an  epizootic  among  its  own 
species,  which  later  spreads  to  other  animals  and  to  man  through 
the  agency  of  fleas,  producing  the  bubonic  form  of  the  disease. 
The  infection  may  then  be  transmitted  from  man  to  man  by 
fomites  and  directly  by  contact,  and  by  infectious  material  sus- 
pended in  the  air,  giving  rise  to  the  pneumonic  form  of  the  dis- 
ease. A  persistent  epizootic  of  chronic  plague  among  rodents 
in  a  new  region  may  give  rise  to  a  new  permanent  endemic 
center. 

In  man  the  disease  occurs  in  two  principal  forms,  the  bubonic 
type,  in  which  the  portal  of  entry  is  on  the  skin  or  mucous  mem- 
brane and  the  disease  is  manifested  by  swelling  of  the  neighboring 
lymph  nodes,  and  the  pneumonic  type  in  which  the  organisms 
are  inhaled  or  aspirated  into  the  lung.  Both  of  these  forms  re- 
sult in  general  bacteremia,  as  a  rule.  The  bubonic  form  is  largely 
due  to  inoculation  of  the  skin  by  bites  of  insects  (fleas),  while  the 
pneumonic  form  is  transmitted  more  directly.  Other  clinical  types 
of  the  disease  occur.  The  death  rate  is  30  to  90  per  cent  in  the 
bubonic  and  98  to  100  per  cent  in  the  pneumonic  type.  In  the 
bacteriological  diagnosis,  the  morphology  of  the  organism  in  the 
tissues  and  in  cultures,  its  effect  upon  rats  and  guinea-pigs,  and, 
finally,  agglutination  of  the  newly  isolated  culture  with  a  known 
immune  pest  serum  are  important  points. 

Immunity,  at  least  a  relative  immunity,  follows  recovery  from 
the  plague.  Artificial  immunity  can  be  induced  by  injection 
of  attenuated  living  cultures  and  by  the  injection  of  killed  bac- 
teria (Haffkine's  method).  Many  modifications  of  the  latter 


THE   BACTERIA   OF    THE    HEMORRHAGIC    SEPTICEMIAS          321 

are  recommended  and  they  constitute  the  practical  method  of 
vaccination  against  plague.  Haffkine  employs  broth  cultures 
incubated  at  25  to  30°  C.  for  six  weeks  under  a  covering  of  sterile 
oil.  The  cultures  are  killed  at  65°  C.,  and  preserved  with  car- 
bolic acid.  The  dose  is  o.i  to  0.5  c.c.  for  children  and  3  to  4  c.c. 
for  an  adult  man.  It  may  be  repeated  after  ten  days.  Good 
results  have  followed  the  use  of  this  prophylactic  in  India. 
Kolle  suspends  two-day  agar  cultures  in  broth  or  salt  solution 
and  kills  at  65°  C.  by  one  to  two  hours  exposure.  Five-tenths  per 
cent  carbolic  acid  is  then  added.  The  dose  injected  is  the  prod- 
uct of  one  agar  culture.  The  vaccination  should  be  taken  by  any 
physician  who  expects  to  handle  plague  bacilli,  even  if  only  in  the 
laboratory. 

Horses  have  been  immunized  by  Yersin,  injecting  first  killed 
bacilli,  later  highly  virulent  bacilli,  and  finally  the  filtrates  of  old 
broth  cultures  intravenously.  The  serum  of  these  horses  in  a 
dose  of  20  c.c.  confers  a  transient  passive  immunity,  and  has 
seemed  to  be  of  value  in  the  treatment  of  a  few  cases  of  plague. 
Its  preparation  is  so  difficult  and  its  potency  so  low  that  it  has 
not  come  into  general  use.  The  serum  has  also  been  injected 
along  with  killed  bacilli  to  confer  immunity  (combined  active 
and  passive  immunization) . 

The  restriction  and  prevention  of  plague  require  measures 
adapted  to  the  special  conditions  existing.  In  general  they  include 
precautions  to  exclude  infected  animals,  wholesale  destruction 
of  rats  and  other  rodents  and  the  artificial  immunization  of  the 
human  population  when  confronted  by  the  disease.  The  eradi- 
cation of  the  endemic  centers  presents  a  problem  of  great  com- 
plexity, requiring  the  recognition  and  destruction  of  the  infected 
species  of  animals. 

Bacillus  (Micrococcus)  melitensis.1 — Bruce  in  1887  dis- 
covered this  organism  in  the  spleen  of  persons  suffering  from  Malta 

1  This  organism  is  classed  as  a  micrococcus  by  most  authors.  It  is  here  classed 
as  a  bacillus  because  of  its  general  resemblance  in  many  of  its  characters  to  B.  pestis. 
None  of  the  Gram-negative  parasitic  cocci  resemble  it  in  respect  to  physiological 
characters  or  in  the  remarkable  ability  to  change  its  host. 

21 


322  SPECIFIC  MICRO-ORGANISMS 

fever  and  obtained  pure  cultures.  Inoculation  of  monkeys  with 
pure  cultures  gives  rise  to  a  disease  resembling  in  detail1  Malta 
fever  in  man. 

The  organism  is  spherical  or  oval  0.3  by  0.4^  in  size,  and  is 
classed  as  a  micrococcus  by  many  bacteriologists.  In  gelatin 
cultures  the  cell  is  somewhat  longer  and  resembles  that  of  a  true 
bacillus.  The  organisms  are  single,  grouped  in  pairs  or  sometimes 
in  short  chains  of  four  to  five  cells.  Capsules  and  spores  have 
not  been  observed.  It  is  non-motile.  Flagella  have  been  de- 
tected by  Gordon  but  other  investigators  have  failed  to  confirm 
the  observation.  The  organism  stains  readily  and  is  Gram- 
negative. 

Cultures  are  obtained  on  ordinary  media  and  growth  is  possi- 
ble between  the  extremes  of  6°  and  45°  C.  The  colonies  develop 
in  one  to  three  days  at  37°  C.  and  are  very  homogeneous.  Gela- 
tin is  not  liquefied  and  neither  gas  nor  acid  is  produced  in  media 
containing  the  various  sugars.  The  organism  is  killed  by  moist 
heat  at  57°  C.  in  10  minutes,  by  dry  heat  at  95°  C.  in  10  minutes 
and  in  i  per  cent  carbolic  acid  in  15  minutes.  It  survives  drying 
for  several  months  and  retains  its  vitality  in  culture  without 
transplantation  for  several  years  if  drying  is  prevented. 

Many  mammals  are  susceptible,  including  guinea-pigs,  rabbits, 
monkeys,  rats  and  mice.  Horses,  cows,  sheep  and  goats  are  not 
only  susceptible  to  inoculation  but  also  contract  the  disease  natu- 
rally. In  all  animals  the  course  of  the  infection  is  usually  chronic 
and  characterized  by  an  irregularly  remittent  fever.  Death  is 
a  common  outcome  in  monkeys.  Often  the  subcutaneous  injec- 
tion or  the  feeding  of  a  minute  quantity  of  the  culture  is  sufficient 
to  infect,  but  for  the  smaller  laboratory  animals  intracerebral  in- 
oculation may  be  necessary. 

Malta  fever  in  man  is  a  chronic  disease  characterized  by  an 

irregularly  remittent  fever.     The  spleen  is  enlarged  and  often 

the  liver  as  well.     Positive  agglutination  of  a  known  culture  of 

B.  melitensis  by  the  patient's  serum  in  dilution  of  i  to  1000  is  an 

1  Eyre  in  Kolle  and  Wassermann,  Handbuch,  1912,  Bd.  IV,  S.  432. 


THE   BACTERIA   OF   THE   HEMORRHAGIC   SEPTICEMIAS         323 

important  aid  in  diagnosis,  and  isolation  of  the  organism  from  the 
circulating  blood,  or  from  the  spleen,  and  its  identification  makes 
the  diagnosis  certain.  Positive  cultures  are  more  often  obtained 
from  the  spleen,  but  the  puncture  of  this  organ  by  the  inexperi- 
enced is  not  without  danger.  Blood  cultures  should  be  made  dur- 
ing a  febrile  period  and  preferably  late  in  the  afternoon.  Death 
occurs  in  i  to  2  per  cent  of  the  cases. 

Careful  investigations  have  shown  that  infection  with  B.  meli- 
tensis  is  endemic  among  the  goats  of  Malta,  from  which  animals 
is  obtained  the  milk  supply  of  the  region.  The  micro-organisms 
are  excreted  in  the  milk.  Monkeys  fed  such  milk  acquire  the 
disease,  and  human  epidemics  of  Malta  fever  have  followed  the 
use  of  such  milk  under  conditions  closely  resembling  those  of 
critical  experimentation.  Other  methods  of  transmission  have 
been  tested  with  negative  results. 

Immunity  follows  recovery  from  the  disease,  but  artificial 
immunization  is  not  yet  a  practical  success. 


CHAPTER  XXII. 

BACTERIACE^E:  THE  COLON,  TYPHOID  AND  DYS- 
ENTERY BACILLI. 

Bacillus  Coli. — This  organism  was  probably  observed  by  sev- 
eral investigators  previous  to  1886  but  it  was  either  neglected  or 
its  significance  was  misinterpreted.  The  first  important  study 
of  it  was  made  by  Escherich  in  that  year,  who  discovered  it  in  the 
feces  of  healthy  infants  and  obtained  it  alone  on  the  aerobic  gela- 
tin plate  cultures  inoculated  with  this  material. 


FIG.  130. — Bacillus  coll  showing  flagella.     (From  McFarland  after  Migula.} 

B.  coli  lives  and  grows  in  the  intestinal  tract  of  man  and  mam- 
mals, and  organisms  closely  resembling  it  have  been  found  in  the 
intestinal  canal  of  other  vertebrates.  It  is  discharged  in  large 
numbers  in  the  feces  and  some  of  these  bacilli  may  continue  their 
growth  in  the  external  world  for  a  time.  The  organism  is  0.4  to 

324 


THE  COLON,  TYPHOID  AND  DYSENTERY  BACILLI      325 

o.7M  wide  and  i  to  6/1  long,  with  rounded  ends,  usually  single  but 
sometimes  occurring  in  threads.  It  is  motile  but  not  very  active^ 
and  many  cells,  even  in  young  cultures,  may  be  motionless. 

There  are  four  to  eight  peritrichous  flagella.  Spores  have 
not  been  observed.  The  bacillus  stains  readily  and  is  Gram- 
negative. 

Cultures  develop  rapidly  at  37°  C.  on  all  ordinary  media. 
The  colony  is  white,  opaque,  often  somewhat  heaped  up  in  the 
center  and  thinner  near  the  edge.  It  may  be  round  with  smooth 
outline  or  the  border  may  be  lobulated.  Under  the  low-power 


FIG.  131. — Bacillus  coli.     Superficial  colony  on  a  gelatin  plate  two  days  old.     X  21. 
(From  McFarland  after  Heim.) 

lens  the  colony  appears  brown,  finely  granular  near  the  periphery 
and  more  coarsely  granular  near  the  center.  It  is  soft  and  moist, 
easily  removed  from  the  medium  and  easily  suspended  as  a  dif- 
fuse cloud  in  water.  Gelatin  is  not  liquefied.  B.  coli  ferments 
dextrose  and  lactose  with  the  production  of  gas  as  well  as  acid. 
It  coagulates  milk  in  24  to  48  hours  at  37°  C.  and  renders  it  acid, 
produces  considerable  indol  in  pepton  solution  and  grows  abun- 
dantly on  potato,  often  producing  a  brown  color. 

Intraperitoneal  injection  of  cultures  into  guinea-pigs  and  rats 
causes  fatal  peritonitis.  Subcutaneous  injection  may  also  cause 
death  but  frequently  results  in  a  local  abscess. 


326  SPECIFIC  MICRO-ORGANISMS 

The  cultures  of  B.  coli  on  ordinary  media  are  practically  free 
from  soluble  poisons,  but  there  is  some  evidence  that  soluble 
poisons  may  be  produced  by  this  organism  under  special  condi- 
tions.1 The  bacterial  cell  substance  is  poisonous. 

As  it  grows  in  the  intestine  the  colon  bacillus  is  a  harmless 
commensal  but  with  a  distinct  tendency  to  invade  the  living 
tissue  and  become  pathogenic  whenever  the  normal  resistance  is 
lowered.  The  bacilli  doubtless  pass  through  the  intestinal  wall 
in  very  small  numbers  during  absorption  of  the  food  and  are  de- 
stroyed in  the  normal  body  fluids  and  tissues  in  a  few  hours.  In 
intestinal  disturbances  the  invasive  properties  and  the  virulence 
are  increased.  In  many  other  regions  of  the  body  the  colon  bacil- 
lus gives  rise  to  inflammation,  often  purulent  in  character.  It 
is  a  common  cause  of  cystitis  and  pyelitis,  and  is  an  important 
agent  in  the  causation  of  peritonitis  following  perforation  of  the 
intestine.  Generalized  infection  with  B.  coli  is  rather  uncommon. 
The  bacilli  frequently  enter  the  blood  stream  from  the  intestine 
during  the  death  agony,  and  are  often  present  in  the  heart's  blood 
at  autopsy,  especially  if  this  is  delayed. 

The  detection  of  B.  coli  in  any  material  is  ordinarily  regarded 
as  evidence  of  fecal  contamination.  Examinations  of  drinking 
water  and  of  shell  liquor  from  oysters  are,  perhaps,  the  most  fre- 
quent applications  of  this  principle.  Fermentation  tubes  of 
dextrose  broth  are  inoculated  with  measured  quantities  of  the 
liquid  to  be  tested,  o.oi  c.c.,  o.i  c.c.  and  i  c.c.  Those  cultures  jn 
which  gas  is  produced  are  plated  on  litmus  lactose  media  and  the 
typical  colonies  transplanted  to  gelatin,  milk,  fermentation  tubes 
of  dextrose_broth  and  agar  slants,  and  for  final  identification  the 
agglutination  test  with  a  known  colon-immune  serum  may  be 
employed. 

Bacillus  (Lactis)  Afe'rogenes. — Escherich  described  this  organ- 
ism in  1886  as  distinct  from  B.  coli.  It  is  non-motile,  is  usually  cap- 
sulated  and  its  colonies  are  thicker  and  less  spreading.  In  other 
respects  it  does  not  differ  materially  from  B.  coli  and  many  authori- 
1  See  Vaughan  and  Novy:  Cellular  Toxins,  Phila.,  1902,  p.  220. 


THE    COLON,    TYPHOID   AND    DYSENTERY  BACILLI 


327 


ties  regard  it  as  a  variety  of  this  species. 
B.  aero  genes  was  found  by  Escherich  in 
the  upper  part  of  the  small  intestine.  It 
is  commonly  present  in  ordinary  cow's 
milk  and  has  been  found  in  the  urine  in 
cystitis1  and  pyelitis. 

Bacillus  (Bacterium)  Pneumoniae.— 
This  organism  was  obtained  by  Fried- 
laender  in  1883  on  gelatin  plates  inocu- 
lated with  material  from  cases  of  pneu- 
monia and  was  confused  by  him  with  the 
organisms  which  he  observed  microscopi- 
cally in  abundance  in  his  material.  The 
latter  were  undoubtedly  pneumococci  (See 
Diplococcus  pneumonia  page  257).  B.  pneu- 
monia is  rather  common  in  the  upper  air 
passages  and  occurs  in  the  lungs  in  some 
cases  of  pneumonia.  It  is  non-motile, 
capsulated  and  Gram-negative,  and  in 
nearly  all  respects  quite  like  B.  aerogenes. 
The  nail-shaped  culture  in  gelatin  stab  is 
regarded  as  specially  typical. 

Bacillus  (Bacterium)  Rhinosclero- 
matis. — This  organism  was  described  by 
von  Frisch  in  1 88  2 .  It  is  readily  obtained, 
often  in  pure  culture,  by  incising  the  lesion 
of  rhinoscleroma  and  spreading  the  blood 
thus  obtained  on  an  agar  surface.2  It  is 
also  found  in  abundance  by  microscopic 
examination  of  sections  of  rhinoscleroma 
tissue.  B.  rhinosderomatis  is  capsulated, 
non-motile  and  in  morphology  and  cultural 

1  Luetscher,  Johns  Hopkins  Hosp.  Bull.,  ion.  Vol 
XXII,  pp.  361-366. 

2  Wright  and  Strong:  New  York  Med.  Journ.,  ion 
Vol.  XCIII,  pp.  516-519. 


FIG.  132. — Friedlan- 
der'spneumobacillus;  gel- 
atin stab  culture,  show- 
ing the  typical  nail-head 
appearance  and  the  for- 
mation of  gas  bubbles,  not 
always  present.  (From 
McFarland  after  Curtis.) 


328  SPECIFIC   MICRO-ORGANISMS 

characters  indistinguishable  from  B.  pneumonia.  It  is  Gram- 
negative  when  stained  by  the  usual  technic.  Its  etiological  rela- 
tion to  rhinoscleroma  is  somewhat  uncertain. 

Rhinoscleroma  is  a  disease  characterized  by  the  occurrence  of 
circumscribed  grayish  nodules  in  the  mucous  membrane  of  the 
nose,  which  tend  slowly  to  extend  without  ulceration.  Histo- 
logically  the  lesion  is  composed  of  granulation  tissue  and  fibrous 
tissue  with  lymphocy tic  infiltration.  Many  of  the  cells  appear 
swollen  and  vacuolated,  so-called  lace-cells,  and  in  and  near  these 
the  bacilli  are  present  in  large  numbers.  The  disease  occurs  in 
Europe  and  has  been  seen  in  a  number  of  Russian  immigrants  to 
the  United  States. 

Bacillus  (Mucosus)  Capsulatus  and  Bacillus  Ozenae  also  occur 
on  the  mucous  membranes  of  the  upper  air  passages.  They  do 
not  appear  to  be  specifically  different  from  B.  pneumonia  of 
Friedlaender. 

Bacillus  Enteritidis. — Gaertner  in  1888  isolated  this  organism 
from  the  spleen  of  a  man  who  died  in  an  epidemic  of  meat  poison- 
ing in  which  57  persons  were  made  ill.  The  meat  was  derived 
from  a  cow,  sick  at  the  time  of  slaughter,  and  this  same  organism 
was  found  in  the  meat  which  had  not  been  sold.  The  bacillus  is 
of  the  same  size  and  shape  as  B.  coli,  but  is  more  actively  motile 
and  has  more  flagella.  It  ferments  dextrose  with  the  production 
of  gas,  does  not  ferment  lactose  nor  coagulate  milk,  nor  does  it 
produce  an  amount  of  indol  appreciable  by  testing  with  sulphuric 
acid  and  nitrite.  Its  cultures  are  highly  toxic,  even  after  they 
have  been  boiled.1  A  typhoid-immune  serum  agglutinates  B. 
enteritidis  in  fairly  high  dilutions.  The  cases  of  food  poisoning 
in  which  it  was  found  were  characterized  by  vomiting  and  diarrhea 
and  at  autopsy  by  severe  enteritis  and  swelling  of  the  lymph 
follicles  of  the  intestine.  Food  poisoning  of  this  type  seems  to 
be  rather  common.2 


1  Vaughan  and  Novy:  Cellular  Toxins,  1902,  p.  207. 

2  Anderson,   Poisoning  from  Bacillus  enteritidis.     The  Military  Surgeon,  1912, 
Vol.  XXXI,  pp.  425-29.     See  also  Marshall's  Microbiology,  1911,  p.  414. 


THE    COLON,    TYPHOID    AND    DYSENTERY   BACILLI  329 

Bacillus  Suipestifer  (B.  Salmonii). — This  organism  occurs  in 
the  intestinal  contents  of  hogs  and  in  the  blood  in  the  late  stages 
of  hog  cholera,  and  was  for  a  time  believed  to  be  the  cause  of  this 
disease.  More  recent  studies  indicate  that  the  etiological  factor 
is  a  filterable  virus  (See  page  373).  B.  suipestifer  resembles  B. 
enteritidis  very  closely. 

Bacillus  Icteroides  was  described  by  Sanarelli  in  1897  as  the 
cause  of  yellow  fever,  a  disease  now  known  to  be  caused  by  a  filter- 
able agent  (page  368).  It  cannot  be  specifically  distinguished 
from  B.  suipestifer. 

Bacillus  Psittacosis  was  found  by  Nocard  in  1892  to  be  the 
cause  of  an  epidemic  pneumonia  transmitted  to  man  from  dis- 
eased parrots.  It  resembles  B.  coli  but  may  be  distinguished  by 
inoculating  parrots,  for  which  it  is  extremely  virulent. 

Bacillus  Typhi  Murium. — L6fHer  in  1890  found  this  organism 
to  be  the  cause  of  a  fatal  epizootic  among  laboratory  mice.  It 
forms  gas  and  acid  from  dextrose,  does  not  produce  indol  nor  co- 
agulate milk.  Mice  are  very  susceptible  and  the  organism  has 
been  employed  as  a  practical  means  of  destroying  mice.  It  seems, 
however,  not  to  be  altogether  harmless  for  larger  animals  and  for 
man,  and  it  is  believed  that  some  of  the  paratyphoid  fever  fol- 
lowing food  poisoning  in  man  has  been  due  to  this  particular 
organism. 

•  Bacillus  (Fsecalis)  Alkaligenes. — This  organism  is  occasionally 
found  in  human  feces  and  is  of  importance  because  of  the  possi- 
bility of  mistaking  it  for  the  typhoid  bacillus,  which  it  resembles 
in  most  respects.  It  does  not  produce  acid  from  any  of  the  sugars 
nor  is  it  agglutinated  by  typhoid  serum.  It  is  not  known  to 
cause  disease. 

Several  other  organisms  of  this  general  type  have  been  found 
in  pathological  conditions  of  man  or  of  animals  and  some  of  them 
have  received  specific  names.  In  certain  irregular  fevers  in  man 
resembling  somewhat  typhoid  fever,  organisms  have  been  found 
in  the  circulating  blood  which  are  agglutinated  by  the  patient's 
serum,  and  which  exhibit  many  of  the  characters  of  the  B.  coli  or 


330 


SPECIFIC   MICRO-ORGANISMS 


B.  enteritidis  groups.  They  are  ordinarily  regarded  as  inter- 
mediate between  B.  coli  and  B.  typhosus  and  are  designated 
as  paracolon  and  paratyphoid  bacilli.  The  diseases  in  which  they 
occur  are  sometimes  traceable  to  meat  poisoning.  B.  enteritidis 
and  B.  typhi  murium  doubtless  occur  in  the  circulating  blood  of 
man  at  times  as  paratyphoid  bacilli.  B.  psittasosis  is  usually 
regarded  as  a  paracolon  bacillus. 

Bacillus  Typhosus. — Eberth  in  1880  and  Koch  in  1880  ob- 
served this  organism  in  the  spleen  and  mesenteric  lymph  glands 


FIG.  133. — Bacillus  of  typhoid  fever.     X  1000. 

of  persons  dying  of  typhoid  fever.  Gaffky  in  1884  obtained  the 
first  pure  cultures.  Metchnikoff1  and  Besredka  in  1911  succeeded 
in  causing  typical  typhoid  fever  in  anthropoid  apes  (chimpanzees) 
by  feeding  them  cultures  of  B.  typhosus,  thus  adding  conclusive 
proof  of  the  causal  relationship  of  this  organism  to  typhoid  fever 
to  the  abundant  strong  evidence  previously  at  hand. 

B.  typhosus  is  found  in  the  intestinal  contents,  mesenteric 
lymph  glands,  spleen,  blood  and  urine  of  patients  suffering  from 
typhoid  fever.  It  is  0.5  to  o.8/t  in  width  and  i  to  4/4  in  length, 

1  Annals  de  VInstltut  Pasteur,  1911,  Vol.  XXV,  193-221. 


THE   COLON,    TYPHOID   AND   DYSENTERY  BACILLI 


331 


commonly  occurring  single  or  in  short  threads,   stains  readily 
with  anilin  dyes  and  is  Gram-negative.     It  is  actively  motile  and 


FIG.  134. — Bacillus  typhosus  showing  flagella.     (After  McFarland.} 

possesses  10  to  20  peritrichous  flagella.     Spores  have  not  been 
observed. 


A  B 

FIG.  135.  —  Colonies  on  gelatin  plate  two  days  old  of  (^4)  Bacillus  typhosus,  and  (B) 
Bacillus  coli.     Y^ZT..     (From  Jordan  after  Heim.) 


The  organism  grows  readily  on  ordinary  media  but  not  so 
luxuriantly  as  B.  coli.     The  colony  is  smaller  but  relatively  more 


33  2  SPECIFIC   M^IO-ORGANISMS 

spread  out  and  thinner  than  that  of  B.  coli,  and  in  semi-solid  media 
the  growth  of  B.  typhosus  may  diffuse  for  quite  a  distance  because 
of  its  active  motility.  Dextrose  is  fermented  with  the  production 
of  acid  but  without  gas.  Lactose  is  not  fermented.  Litmus 
milk  is  rendered  slightly  acid  and  later  becomes  alkaline  without 
coagulation.  On  potato  the  growth  is  almost  invisible.  In  Dun- 
ham's pepton-salt  solution,  indol  is  not  produced  in  sufficiently 
large  quantities  to  be  detected,  but  indol  can  be  demonstrated 
in  old  cultures  in  5  per  cent  pepton.  Growth  is  most  rapid  at 
37°~39°  C.,  but  occurs  also  at  room  temperature. 

B.  typhosus  is  killed  by  moist  heat  in  10  to  15  minutes,  and  by 
5  per  cent  carbolic  acid  or  i-iooo  mercuric  chloride  in  three  to 
five  minutes,  when  exposed  in  aqueous  suspension.  It  resists  dry- 
ing for  several  days  and  may  be  alive  in  dry  dust.  The  longevity 
of  B.  typhosus  in  surface  waters  has  been  studied  by  several  in- 
vestigators without  full  agreement.  In  general  B.  typhosus  would 
seem  to  survive  in  such  water  only  for  three  to  ten  days  except 
it  be  taken  up  by  aquatic  animals,  such  as  the  shellfish,  when  it 
may  persist  for  several  weeks.  In  soil  and  in  frozen  material 
the  bacillus  may  live  a  much  longer  time.  Freezing  and  thawing 
destroys  a  large  percentage  of  the  bacilli  in  a  given  liquid  but 
does  not  destroy  them  all. 

The  poisons  are  intimately  associated  with  the  cell  substance, 
and  it  is  not  often  that  culture  filtrates  are  found  to  be  toxic. 
The  dead  germ  substance  is  somewhat  poisonous,  and  when  it 
is  disintegrated  by  physical  comminution  or  by  digestion  with 
dilute  alkali  at  a  high  temperature,  or  by  the  action  of  serum1 
upon  it,  there  are  set  free  quite  powerful  poisons  or  perhaps  differ- 
ent quantities  of  the  same  poison. 

The  various  small  laboratory  animals  are  very  susceptible  to 
intraperitoneal  inoculation  with  B.  typhosus  and  usually  die  in 
24  to  48  hours  with  acute  peritonitis  and  bacteremia.  The  dis- 
ease produced  bears  no  resemblance  to  typhoid  fever  in  man. 
In  chimpanzees  a  very  typical  attack  of  typhoid  fever  has  been 
1  Zinsser:  Journ.  Exp.  Med.,  1913,  Vol.  XVII,  pp.  117-131. 


THE  COLON,  TYPHOID  AND  DYSENTERY  BACILLI      333 

caused  by  feeding  the  organisms,  with  resulting  lesions  in  the  in- 
testine, bacilli  in  the  blood  and  spleen,  and  a  continued  fever. 

Typhoid  fever  exists  generally  throughout  the  temperate 
zone,  is  present  throughout  the  year  but  most  prevalent  in  the 
fall.  The  usual  mode  of  infection  is  undoubtedly  through  food 
and  drink.  The  bacilli  swallowed  survive  in  part  the  action  of 
the  gastric  juice  and  so  gain  the  lumen  of  the  duodenum.  The 
first  multiplication  seems  to  occur  here1  in  a  location  fairly  free 
from  bacteria  in  health.  The  infection  extends  along  the  wall 
of  the  intestine,  involving  especially  the  lymphatic  structures, 
solitary  glands  and  Peyer's  patches.  The  bacteria  pass  into 
the  lymph  stream  to  be  carried  to  the  mesenteric  nodes,  spleen 
and  into  the  blood.  At  the  onset  of  definite  symptoms  of  typhoid 
fever  the  bacilli  have  usually  reached  the  general  blood  circu- 
lation. Subsequently  the  infection  reaches  the  gall  bladder,  per- 
haps by  extension  along  the  common  bile  duct  and  cystic  duct  or 
perhaps  by  the  blood  stream  through  the  liver;  the  organisms 
also  pass  through  the  kidney  and  multiply  in  the  contents  of  the 
urinary  bladder.  They  are  present  in  the  rose  spots  on  the 
skin.  The  bacilli  are  often  present  in  the  feces  in  small  numbers, 
the  abundance  of  other  organisms  making  their  isolation  and 
recognition  difficult.  At  times  localized  inflammations  due  to 
B.  typhosus  develop  elsewhere  in  the  body,  as  in  the  lungs.  It 
is  evident  therefore  that  the  bacilli  may  leave  the  body  of  the 
patient  through  many  channels,  but  chiefly  with  the  urine  and 
feces.  Even  after  recovery  the  patient  may  continue  to  dis- 
charge virulent  bacilli  for  months  or  years.  It  is  estimated  that 
one  per  cent  of  recovered  cases  are  persistent  carriers  of  the  in- 
fectious agent. 

The  bacteriological  diagnosis  of  typhoid  fever  depends  upon 
isolation  and  recognition  of  the  germ  or  detection  of  specific  sub- 
stances in  the  blood  produced  by  the  patient  as  a  reaction  to  the 
presence  of  B.  typhosus.  B.  typhosus  is  sought  by  blood  culture 
(see  page  101)  diluting  the  blood  with  large  amounts  of  broth 
1  Hess:  Journ.  Infect,  Diseases,  1912,  Vol.  XI,  pp.  71-76. 


334  SPECIFIC  MICRO-ORGANISMS 

(200  c.c.  of  broth  to  2  c.c.  of  blood)  as  well  as  inoculating  tubes  of 
bile  and  the  usual  agar  plates;  by  cultures  from  the  rose  spots, 
and  by  cultures  inoculated  with  duodenal  fluid.  These  methods 
are  likely  to  be  successful  very  early  in  the  disease.  Later  it 
is  well  to  make  cultural  examination  of  the  feces  and  urine, 
especially  just  before  discharging  a  recovered  patient. 

The  detection  of  B.  typhosus  in  feces  requires  special  care. 
Russell  recommends  plating  the  feces  on  Endo's  medium,1  fishing 
of  the  promising  colonies  to  a  slant  of  his  double-sugar  medium,2 
inoculating  both  as  a  streak  and  stab,  and  then  making  the  agglu- 
tination test  with  known  serum  upon  the  typical  cultures  in  the 
double-sugar  medium.  The  examination  is  thus  completed  in 
two  or  three  days. 

The  specific  antibody  ordinarily  sought  in  the  blood  is  the 
typhoid  agglutinin.  A  few  drops  of  blood  in  a  Wright's  capsule 
suffice  for  the  microscopic  test  (see  page  211).  A  young  active 
culture  (broth  three  hours)  of  a  known  B.  typhosus  is  used,  and 
the  serum  is  tested  in  dilutions  of  1:20,  1:40  and  1:80,  observed 
for  an  hour.  Normal  serum  rarely  shows  any  clumping  in  any 
of  these  dilutions  at  the  end  of  an  hour.  This  agglutination  test 
is  of  little  or  no  value  if  the  patient  has  received  typhoid  vaccine 
within  a  year. 

Transmission  of  the  disease  takes  place  in  a  variety  of  ways. 
To  the  best  of  our  knowledge,  the  typhoid  bacilli  come  only  from 
human  individuals  infected  with  them.  Some  of  these  actually 

1  For  Endo's  medium  a  stiff  lactose  agar  is  prepared  containing  Liebig's  extract 
5  grams,  salt  5  grams,  pepton  10  grams,  lactose  10  grams  and  agar  30  grams  in  1000 
c.c.  of  water.     This  is  sterilized  in  flasks  containing  100  c.c.  each.     When  needed 
the  contents  of  a  flask  is  liquefied,  enough  sodium  hydroxide  is  added  [to  make  the 
reaction  0.2  per  cent  acid  to  phenolphthalein  and  to  it  are  then  added  10  drops  of 
saturated  alcoholic  solution  of  basic  fuchsin,  and  20  drops  of  a  freshly  prepared  solu- 
tion of  sodium  sulphite.     The  material  is  well  mixed  and  poured  into  8  or  10  Petri 
dishes,  allowed  to  solidify  and  dried  in  the  incubator  to  remove  water  from  the  sur- 
face before  use.     Fecal  material  is  spread  by  means  of  a  bent  glass  rod  over  the  sur- 
face of  several  plates  in  succession. 

2  The  double-sugar  medium  is  a  2  to  3  per  cent  agar,  neutral  to  litmus,  to  which 
has  been  added  i  per  cent  lactose  and  o.i  per  cent  glucose.    On  this  medium  B. 
typhosus  does  not  change  the  color  when  it  is  growing  on  the  surface,  but  produces 
a  red  (acid)  color  about  the  stab.     See  Russell,  Journ.  Med.  Rsch.,  1911,  Vol.  XX, 
pp.  217-229. 


THE   COLON,    TYPHOID   AND   DYSENTERY  BACILLI  335 

suffer  from  typhoid  fever,  while  others  are  merely  healthy  carriers 
of  the  infection.  From  them  as  centers  the  bacilli  are  distributed 
by  contact  and  by  intermediate  objects.  B.  typhosus  is  able  to 
live  for  a  considerable  time  in  the  external  world,  probably  for 
one  to  three  weeks  in  ordinary  surface  waters  and  longer  in  soil. 
It  is  able  to  grow  and  multiply  in  some  foods,  especially  milk. 
Water  supplies  contaminated  with  feces  and  urine  from  patients 
or  from  healthy  carriers  have  unquestionably  been  an  important 
factor  in  the  causation  of  typhoid  fever  in  the  past,  and  the  pro- 
vision of  a  supply  of  drinking  water  free  from  all  suspicion  of 
recent  mixture  with  sewage  is  the  first  step  in  the  control  of  this 
disease  in  a  community.  The  infected  oyster  from  a  sewage- 
polluted  oyster  bed  is  another  source  of  typhoid  fever.  The 
contamination  of  food  by  permanent  carriers  of  the  bacilli  is 
difficult  to  control.  All  possible  means  need  to  be  employed  to 
prevent  these  persons  from  handling  foods  prepared  for  consump- 
tion, and  especially  milk.  Flies  (Musca  domestica)  are  important 
aids  in  the  transfer  of  bacilli  from  discharges  containing  them, 
especially  from  open  privies,  to  foods  exposed  for  sale  or  being 
prepared  in  neighboring  unscreened  kitchens,  i  i 

The  prevention  of  typhoid  fever  by  restricting  the  distribu- 
tion of  the  bacilli  has  been  only  partially  successful  in  civil  life 
and  in  armies  on  a  war  footing  it  has  proven  wholly  ineffective. 
Vaccination  to  prevent  typhoid  fever  was  first  extensively  prac- 
tised by  Wright  in  the  British  army.  Russell1  following  the  method 
developed  by  Wright  and  Leishman  has  prepared  a  vaccine  with 
which  practically  the  whole  U.  S.  army  has  been  inoculated. 
The  vaccine  is  a  suspension  of  B.  typhosus  in  salt  solution,  stand- 
ardized by  microscopic  count  of  the  bacterial  cells,  sterilized  by 
heating  at  53°  to  56°  for  an  hour  and  preserved  by  the  addition 
of  0.25  per  cent  trikersol.  Three  injections  are  given  subcutane- 
ously  at  intervals  of  10  days,  500  million  bacilli  at  the  first  dose 
and  1000  million  at  each  of  the  following  doses.  The  results 

1  Russell:  Boston  Med.  and  Surg.  Journ.,  1911,  Vol.  CLXIV,  pp.  1-8;  Harvey 
Lecture,  1913. 


336  SPECIFIC   MICRO-ORGANISMS 

in  the  U.  S.  army  have  been  remarkably  good,  rivaling  those 
obtained  with  the  use  of  vaccinia  in  the  prevention  of  small-pox. 

Bacillus  (Bacterium)  Dysenteriae. — Shiga  in  1898  isolated 
this  organism  from  the  feces  of  patients  suffering  from  dysentery, 
showed  that  it  is  agglutinated  by  the  blood  of  dysenteric  patients 
in  high  dilutions  and  not  by  normal  human  blood. 

B.  dysenteries  is  about  o.6/x  in  width  by  2  to  4/4  in  length,  usually 
single  and  non-motile.  It  stains  readily  and  is  Gram-negative. 
Involution  forms  are  common  in  older  cultures.  The  organism 
grows  readily  on  ordinary  media  and  its  cultures  resemble  those 
of  B.  typhosus  very  closely.  Gelatin  is  not  liquefied;  no  indol  is 
produced  in  pep  ton  solution;  no  gas  is  formed  from  any  of  the 
sugars;  milk  is  rendered  slightly  acid  and  then  alkaline  without 
coagulation.  It  differs  from  the  typhoid  bacillus  in  failing  to 
ferment  mannite  and  maltose. 

When  cultures  are  injected  intravenously  into  rabbits  severe 
diarrhea  is  produced,  which  may  be  bloody.  The  animal  usually 
dies  in  a  few  days,  and  if  it  recovers  often  exhibits  paralysis  of 
the  hind  legs.  Similar  results  are  obtained  by  the  injection  of 
dead  bacilli,  indicating  that  the  effect  is  toxic  rather  than  infec- 
tious. Kittens  and  puppies  have  been  infected  by  introducing 
dysentery  bacilli  into  the  stomach,  resulting  in  diarrhea  with  the 
intestinal  lesions  of  dysentery.  The  toxins  seem  to  be  intimately 
bound  up  in  the  cells  in  young  cultures,  but  readily  set  free  into 
solution  after  the  bacilli  are  killed.  Culture  filtrates,  of  which 
0.02  c.c.  suffices  to  kill  a  rabbit  in  24  hours,  have  been  obtained. 

Acute  epidemic  dysentery  is  the  disease  in  which  this  organism 
is  found.  The  infectious  agent  is  found  on  the  membrane  of  the 
large  intestine,  which  is  diffusely  inflamed,  often  covered  with  a 
fibrinous  exudate,  or  by  a  pseudo-membrane.  Later  numerous 
ulcers  may  be  found.  The  bacilli  are  only  very  rarely  found  in 
the  blood  or  internal  organs.  The  blood  of  the  patient  aggluti- 
nates the  bacillus  of  Shiga  in  dilutions  of  i  to  50  or  i  to  100.  The 
mortality  is  about  25  per  cent,  but  variable  in  different  epidemics. 

Horses  have  been  immunized  with  cultures  of  B.  dysenteric  and 


THE  COLON,  TYPHOID  AND  DYSENTERY  BACILLI      337 

the  serum  of  these  animals  has  been  found  to  be  antitoxic  as  well  as 
bactericidal.     Its  use  in  treatment  has  given  promising  results  and_ 
seems  to  cause  a  reduction  in  the  death  rate  of  about  50  per  cent. 

Paradysentery  Bacilli. — Flexner  in  1899  isolated  a  bacillus 
from  cases  of  dysentery  in  the  Philippines  which  at  the  time  was 
considered  to  be  the  same  as  the  Shiga  bacillus.  Kruse,  although 
he  found  the  Shiga  bacillus  in  epidemic  dysentery,  found  a  some- 
what different  organism  in  " asylum  dysentery"  or  pseudo- 
dysentery,  which  proved  to  be  identical  with  the  Flexner  bacillus. 
Between  1901  and  1903  a  number  of  strains  of  bacilli  resembling 
somewhat  B.  dysenteries  were  isolated  -by  different  investigators 
from  epidemics  of  diarrheal  disorder,  especially  in  the  Eastern 
United  States.  The  paradysentery  bacilli  are  indistinguishable 
from  B.  dysenteries  in  morphology  or  in  cultures  on  ordinary 
media.  They  are  all  much  less  toxic  to  rabbits  than  the  Shiga 
bacillus,  and  they  all  ferment  mannite  with  the  production  of 
acid,  while  the  Shiga  bacillus  does  not. 

The  bacteria  considered  in  this  chapter  are  all  inhabitants 
of  the  alimentary  canal  (mouth,  pharynx,  intestine)  of  man  or 
other  mammals.  They  are  small  bacilli,  Gram-negative,  without 
spores  and  without  the  ability  to  liquefy  gelatin.  They  vary 
from  each  other  in  motility,  possession  of  flagella,  possession  of 
capsules,  and  in  their  ability  to  form  poisonous  substances  and 
to  ferment  various  carbohydrates.  Media  containing  various 
carbohydrates  along  with  an  indicator  such  as  litmus  to  show 
the  production  of  acid,  and  contained  in  fermentation  tubes  so 
as  to  measure  the  production  of  gas,  are  very  useful  in  differentiat- 
ing1 the  various  types  of  bacteria  in  this  group.  Thus,  in  a 

1  Hiss  has  devised  a  very  useful  medium  for  this  purpose  which  obviates  the  neces- 
sity of  using  the  fermentation  tube  to  detect  the  gas.  His  serum- water  medium  is 
made  by  mixing  beef  serum,  i  part,  with  distilled  water,  2  to  3  parts,  and  steaming 
15  minutes  to  destroy  enzymes.  Pure  litmus  solution  (about  i  part  of  a  5  per  cent 
solution  to  100  parts  of  the  medium)  is  then  added  to  produce  a  deep  blue  color. 
The  medium  is  divided  into  several  portions  and  i  per  cent  of  the  desired  carbo- 
hydrate is  added  to  its  respective  portion.  The  sugar  serum-water  media  are  then 
sterilized  at  100°  C.,  on  three  days.  Fermentation  is  shown  not  only  by  the  redden- 
ing of  the  litmus  but  also  by  coagulation  of  the  liquid  medium,  and  gas  production 
is  shown  by  bubbles  caught  in  the  coagulum.  (Hiss  and  Zinsser:  Text-book  of 
Bacteriology,  1910,  p.  132.) 


SPECIFIC  MICRO-ORGANISMS 

broth  containing  maltose,  B.  typhosus  produces  acid,  B.  coli 
produces  acid  and  gas,  and  B.  dysenteries  produces  neither. 
Specific  agglutination  with  the  serum  of  an  animal  immunized 
with  a  known  culture  constitutes  the  most  important  test  in  the 
identification  of  unknown  forms  falling  within  this  group.  This 
test  may  be  used  with  the  capsulated  species  after  they  have 
lost  the  tendency  to  form  capsules  through  propagation  on  artifi- 
cial media. 1 

1  Fitzgerald:  Proc.  Soc.  Biol.  and  Med.,  1913,  Vol.  X,  pp.  52-53 


CHAPTER  XXIII. 

BACTERIACE^:   BACILLUS    MALLEI   AND    MISCELLA- 
NEOUS BACILLI. 

Bacillus  (Bacterium)  Mallei. — Loffler  and  Schiitz  in  1882 
obtained  pure  cultures  of  this  organism  from  glandered  horses 
and  produced  glanders  by  the  injection  of  these  pure  cultures. 

The  bacillus  is  0.3  to  o.5ju  wide  and  2  to  5/4  long,  usually 
straight  with  rounded  ends,  but  sometimes  irregular  in  shape. 
Filamentous  and  branched  forms  have  been  observed  in  cultures. 


FIG.  136. — Bacillus    mallei  from  an    agar    culture. 

Williams.) 


X     1 1  oo.     (After  Park   and 


It  is  not  motile.  Spores  have  not  been  observed.  B.  mallei  is 
stained  with  moderate  difficulty  and  often  stains  unevenly  like 
the  tubercle  and  diphtheria  bacilli.  After  being  stained,  the 
bacterium  is  easily  decolorized  in  weak  acid  or  alcohol;  it  is  also 
Gram-negative.  Cultures  develop  on  ordinary  media,  better  on 
glycerinated  media,  at  temperatures  ranging  from  22 

339 


0  to  42°  C., 


340  SPECIFIC   MICRO-ORGANISMS 

best  at  37°  C.  On  potato  at  37°  C.  a  viscid  yellowish-brown 
growth  develops  surrounded  by  a  greenish  stain  on  the  potato. 
Gelatin  is  not  liquefied.  The  organism  is  killed  by  moist  heat 
at  55°  C.  in  10  minutes,  and  in  2  to  5  minutes  by  5  per  cent 
carbolic  acid  or  i  to  1000  mercuric  chloride.  It  survives  drying 
for  only  a  few  weeks  and  dies  out  quickly  in  water.  Many 
mammals  are  susceptible  to  inoculation,  including  horses,  guinea- 
pigs,  cats  and  dogs.  Cattle  are  immune.  Man  is  susceptible 
and  human  glanders  frequently  ends  in  death. 

Mallein  is  analogous  to  tuberculin.  A  culture  in  glycerin 
broth  incubated  for  six  weeks  is  steamed  and  filtered,  and  the 
filtrate  evaporated  to  one-tenth  the  original  volume  is  the  mallein. 
This  substance  is  toxic  to  animals  suffering  from  glanders  but 
not  poisonous  to  healthy  animals. 

Glanders  is  a  disease  most  common  in  horses,  mules  and  asses. 
It  begins  as  an  inflammation  of  the  nasal  mucosa  with  localized 
nodular  infiltrations  which  later  ulcerate.  The  infection  may 
become  generalized  at  once  causing  acute  glanders  and  death  in 
one  to  six  weeks,  or  it  may  progress  very  slowly  and  persist  for 
years  as  chronic  glanders.  The  chronic  type  is  common  in  horses. 
After  apparent  recovery  from  the  disease  nodules  containing 
living  bacilli  may  be  found  in  the  lungs.  Histologically  the  gland- 
ers nodule  consists  of  granulation  tissue  infiltrated  with  leukocytes 
and  tending  to  become  purulent  at  the  center.  The  bacilli  leave 
the  body  in  the  nasal  secretion  and  in  the  discharge  from  ulcers. 
Infection  of  equines  takes  place  most  frequently  by  ingestion  of 
food  soiled  by  these  discharges.  In  man  the  disease  seems  to 
result  from  inoculation  of  small  wounds  in  the  skin.  It  often 
runs  an  acute  course  terminating  in  death,  but  chronic  glanders 
with  recovery  also  occurs  in  man.  A  few  sad  laboratory  accidents 
in  which  workers  have  become  inoculated  with  glanders  have 
emphasized  the  necessity  for  caution  in  handling  this  organism. 

The  bacteriological  diagnosis  depends  upon  (i)  identification 
of  B.  mallei,  (2)  reaction  of  the  animal  to  mallein,  (3)  agglutina- 
tion reaction,  and  (4)  complement  fixation.  For  the  recognition 


BACILLUS    MALLEI    AND    MISCELLANEOUS   BACILLI  341 

of  the  bacillus,  some  of  the  suspected  material  is  suspended  in 
broth  and  injected  into  the  peritoneal  cavity  of  a  male  guinea-pig^ 
(method  of  Straus).  If  B.  mallei  is  present  a  general  inflamma- 
tion of  the  peritoneum  develops  and  after  three  or  four  days  the 
testicles  of  the  animal  become  swollen,  inflamed  and  later  suppu- 
rate. They  may  burst  through  the  scrotum.  Cultures  should 
be  made  from  this  pus  on  plates  of  glycerin  agar  and  the  colonies 
transplanted  to  potato  at  37°  C.  Very  few  other  organisms 
give  rise  to  a  similar  pathological  picture  in  the  guinea-pig.  At 
the  same  time  the  mallein  test  is  carried  out  by  injecting  0.2  c.c. 
of  the  concentrated  mallein  diluted  with  0.25  per  cent  solution 
of  carbolic  acid  into  the  suspected  horse.  The  presence  of  gland- 
ers is  indicated  by  a  rise  in  temperature  of  2°  to  5°  F.,  signs  of 
general  intoxication,  and  especially  by  swelling  and  inflammation 
at  the  site  of  injection.  For  the  agglutination  test  the  serum 
is  diluted  to  i :  500  to  i :  3000.  Positive  results  with  lower  dilu- 
tions may  apparently  be  given  by  normal  horses.  The  comple- 
ment-fixation test  follows  the  principles  of  Wassermann  test  for 
syphilis,  a  culture  of  B.  mallei  being  employed  as  antigen.1  At- 
tempts at  immunization  have  not  been  practically  successful. 

Bacillus  (Bacterium)  Abortus. — Bang  and  Stribolt  isolated 
this  organism  from  the  uterus  of  a  cow  suffering  from  the  disease 
known  as  contagious  abortion,  and  reproduced  the  disease  by  in- 
oculating healthy  cows  with  these  cultures.  The  organism  is 
of  interest  because  of  its  behavior  toward  oxygen  when  first  iso- 
lated. It  fails  to  grow  in  the  air  or  in  hydrogen,  but  grows  in 
a  partial  pressure  of  oxygen  somewhat  below  that  of  the  atmos- 
phere. The  bacillus  is  pathogenic  for  a  number  of  different 
mammals,  and  in  guinea-pigs  it  causes  granulomatous  lesions 
resembling  somewhat  those  of  tuberculosis.2  The  organism 
occurs  rather  frequently  in  market  milk.  It  is  not  known  to 
infect  man. 

1  Mohler  and  Eichorn:  Twenty-seventh  Annual  Rep.  Bur.  Anim.  Industry,  U.  S. 
Dept.  Agr.,  1910;  reprinted  as  Circular  191  (1912). 

2  Smith  and  Fabyan:  Centr.  f.  Bakt.,  I,  Abt.  Orig.,  1912,  Bd.  LXI,  S.  549-555. 
Fabyan,  Journ.  Med.  Rsch.,  1912,  Vol.  XXV,  p.  441-488. 


342  SPECIFIC  MICRO-ORGANISMS 

Bacillus  (Bacterium)  Acne. — This  minute  non-motile  organ- 
ism, first  described  by  Gilchrist,  is  constantly  present  in  the  pap- 
ules and  pustules  of  the  common  skin  affection,  acne  vulgaris. 
Cultures  are  most  readily  obtained  by  expressing,  with  careful 
asepsis,  some  of  the  cheesy  pus  from  a  recent  papule  and  mixing 
it  with  2  c.c.  of  ascitic  fluid  in  a  test-tube.  Dilutions  from  this 
are  made  to  similar  amounts  of  ascitic  fluid  in  series  (about  five 
tubes  in  all).  To  each  tube  are  then  added  8  c.c.  of  melted 
glucose  agar  cooled  to  50°  C.,  the  contents  of  each  tube  mixed 
without  introducing  air  bubbles  and  then  quickly  solidified  by 
immersion  in  cold  water.  The  colonies  of  B.  acne  develop  at 
37°  C.  after  five  to  ten  days,  beginning  about  8  mm.  beneath 
the  surface,  and  they  grow  best  in  a  narrow  zone  about  5  mm.  in 
depth.  The  colonies  attain  a  large  size  (3  mm.)  and  an  abundant 
supply  of  bacillary  substance  for  preparation  of  vaccine  may  be 
obtained  by  thrusting  a  sterile  glass  capillary  into  such  a  colony. 
In  its  behavior  to  oxygen  when  first  isolated  the  organism  exhibits 
the  same  peculiarity  as  the  bacillus  mentioned  in  the  preceding 
paragraph. 

Bacillus  (Bacterium)  Bifidus. — Tissier  in  1898  showed  that 
the  Gram-positive  bacillus  predominant  in  the  stools  of  healthy 
nurslings  is  not  a  form  of  B.  coll  as  had  been  supposed  since  the 
investigations  of  Escherich  (1886)  but  is  an  entirely  different 
organism.  He  obtained  cultures  by  making  a  series  of  dilutions 
(five  to  ten  tubes)  in  tall  tubes  of  glucose  agar  by  the  method  of 
Veillon  (see  page  112).  The  colonies  develop  best  about  i  to  2 
cm.  beneath  the  surface  after  three  to  eight  days  at  37°  C.  In 
these  colonies  many  of  the  bacilli  show  dichotomous  branching. 
Bifid  forms  are  also  sometimes  seen  in  stools  and  in  mixed  cul- 
tures in  broth.  The  organism  produces  a  strong  acid  reaction 
and  the  cultures  soon  die  out.  The  bifid  forms  are  doubtless 
involutions  due  to  presence  of  unfavorable  amounts  of  acid. 

Bacillus  (Bacterium)  Bulgaricus. — This  organism  is  a  rather 
large  rod  i  by  6ju  approximately.  It  occurs  in  milk  and  milk 
products  and  is  especially  abundant  in  milk  fermented  at  40°  C. 


BACILLUS  MALLEI  AND  MISCELLANEOUS  BACILLI      343 

for  three  or  four  days.  Colonies  may  be  obtained  on  plates  of 
milk  agar  (i  12)  incubated  at  37°  C.  in  hydrogen.  A  high  degree 
of  acidity  (lactic  acid)  is  produced  in  the  cultures  of  this  organism, 
and  it  is  employed  to  some  extent  in  the  preparation  of  acid-milk 
beverages. 

Bacillus  (Proteus)  Vulgaris. — Hauser  in  1885  discovered 
this  organism  in  putrefying  infusions  of  animal  matter.  It  is  an 
actively  motile  rod  0.6 n  in  thickness  and  exceedingly  variable  in 
length,  with  abundant  flagella.  Spores  have  not  been  observed. 
It  is  universally  distributed  in  the  soil  and  is  abundant  in  putrefy- 
ing flesh.  Gelatin  is  rapidly  liquefied.  Food  poisoning  in  man 
has  been  ascribed  to  this  organism.  It  is  also  capable  of  infecting 
laboratory  animals  when  injected  in  large  doses. 

Bacillus  Pyocyaneus  (Pseudomonas  Pyocyanea). — Gessard 
in  1882  isolated  this  organism  from  green  pus.  It  is  a  slender 
rod,  actively  motile.  A  soluble  blue-green  pigment  is  produced 
in  the  cultures.  Gelatin  is  liquefied.  Guinea-pigs  are  susceptible 
to  intraperitoneal  inoculation.  In  man  the  organism  is  most 
common  in  the  pus  from  wounds,  where  its  presence  is  considered 
as  only  mildly  deleterious.  The  bacillus  has  also  been  found  in 
otitis  media  and  a  few  cases  of  fatal  generalized  infection  with  B. 
pyocyaneits  have  been  described. 

Bacillus  Fluorescens  var.  Putidus. — This  non-pathogenic 
actively  motile  rod  is  common  in  putrefying  material.  It  pro- 
duces spores  when  grown  on  quince  jelly.  The  greenish-yellow 
pigment  is  soluble  in  water.  Gelatin  is  not  liquefied.  A  number 
of  different  fluorescing  bacilli  have  been  found  in  the  soil  and 
surface  waters.  Some  of  them  liquefy  gelatin. 

Bacillus  Violaceus. — This  is  a  non-pathogenic  water  bacterium 
which  produces  a  pigment  of  deep  violet  color.  It  is  actively 
motile  and  liquefies  gelatin  rapidly.  The  pigment  is  not  soluble 
in  water.  Several  different  bacteria  are  known  which  produce 
a  violet  pigment. 

Bacillus  Cyanogenus  (Pseudomonas  Syncyanea). — This  non- 
pathogenic  actively  motile  organism  produces  a  bluish-black 


344  SPECIFIC   MICRO-ORGANISMS 

pigment  which  is  soluble  in  water.  Gelatin  is  not  liquefied. 
B.  cyanogenus  sometimes  causes  trouble  in  dairies  as  its  growth 
in  milk  imparts  a  blue  color  to  it. 

Bacillus  Prodigiosus. — This  small  oval  organism  grows  rapidly 
at  room  temperature  on  ordinary  media  and  is  occasionally 
observed  on  foodstuffs  such  as  moist  bread  and  potatoes.  Ordi- 
narily it  is  encapsulated  and  non-motile,  but  it  sometimes  possesses 
flagella.  Gelatin  is  rapidly  liquefied.  A  red  pigment  is  produced 
at  room  temperature  but  not  at  37°  C.  This  pigment  is  insoluble 
in  water.  Large  doses  of  B.  prodigiosus  injected  into  animals 
sometimes  gives  rise  to  signs  of  intoxication. 


CHAPTER  XXIV. 
SPIRZLLACE^E  AND  THE  DISEASES  CAUSED  BY  THEM. 

Spirillum  Rubrum. — Esmarch  discovered  this  organism  in 
the  body  of  a  dead  mouse.  It  is  of  chief  interest  as  a  harmless 
example  of  spiral  bacterium  for  class  study.  It  grows  rather 
slowly  at  room  temperature  without  liquefying  gelatin.  A  dull 
red  pigment,  insoluble  in  water,  is  produced  even  in  the  absence 
of  oxygen.  Growth  occurs  at  37°  and  also  in  the  refrigerator  at 
5°  to  10°  C.  When  grown  at  temperatures  above  20°  C.  the 
organism  is  a  relatively  short,  slightly  bent  rod  and  its  spiral 
nature  is  not  very  evident.  At  10°  C.  beautiful  long  spirals  are 
produced  in  broth  cultures.  It  is  actively  motile. 

Spirillum  Cholerae  (Microspira  Comma). — Koch  in  1883 
discovered  this  organism  in  the  intestinal  discharges  of  patients 
suffering  from  Asiatic  cholera,  and  continuing  his  studies  in  India 
in  the  same  year  established  this  organism  as  the  probable  cause 
of  cholera.  It  occurs  in  the  intestinal  contents  and  feces  of  cholera 
patients,  often  in  great  abundance,  rarely  in  the  feces  of  healthy 
persons,  and  has  been  found  at  times  in  surface  waters,  and  in 
drinking  water  during  epidemics  of  cholera. 

Sp.  cholera  is  a  curved  cylinder  about  0.4 /x  in  thickness  and 
i.5/z  in  length.  In  older  cultures  in  broth  long  spiral  forms  occur. 
There  is  considerable  variation  in  shape  in  cultures  older  than 
48  h,ours.  The  organism  is  actively  motile  and  possesses  a  single 
flagellum  at  one  end.  Those  short  spirals  showing  more  than 
one  flagellum  are  not  to  be  regarded  as  true  cholera  germs. 
Spores  have  not  been  observed.  The  spirillum  stains  readily 
and  is  Gram-negative. 

It  grows  well  and  rapidly  on  ordinary  media.  The  reaction 
needs  to  be  distinctly  alkaline  to  litmus  as  the  organism  is  very 

345 


346  SPECIFIC  MICRO-ORGANISMS 

sensitive  to  acids.  Colonies  appear  on  gelatin  at  22°  C.  in  about 
24  hours  as  circular  disks  with  somewhat  irregular  border  and 
granular  interior.  A  few  hours  later  the  gelatin  begins  to  liquefy. 
In  pep  ton-salt  solution  both  indol  and  nitrite  are  formed,  so  that 
the  addition  of  sulphuric  acid  gives  rise  to  the  red  color  due  to 
nitroso-indol.  This  has  been  called  the  cholera-red  reaction,  but 
it  is  of  course  not  a  specific  test  for  this  organism.  In  milk  there 
occurs  abundant  growth  without  apparent  change  in  the  medium. 
In  broth,  growth  is  extremely  rapid  and  a  pellicle  forms  in  24 


FIG.  137. — Cholera  vibrios,  short  forms.     (From  Kolle  and  Schurmann  after  Zettnow.') 

hours.  The  rapid  growth  in  pepton  solution  (pepton  i  per  cent, 
salt  0.5  per  cent)  and  the  tendency  for  the  organisms  to  collect 
at  the  surface  are  utilized  in  practical  enrichment  for  purposes 
of  diagnosis.  The  spirillum  is  an  obligate  aerobe.  It  is  very 
easily  killed.  If  dried  on  a  cover-glass  at  37°  C.,  the  organisms 
are  all  dead  in  two  hours.  It  seems  impossible,  therefore,  for  the 
infection  to  be  distributed  in  dry  dust.  Moist  heat  at  56°  C. 
kills  the  cholera  spirilla  in  30  minutes.  They  are  also  easily 
killed  by  chemical  germicides.  Milk  of  lime  is  recommended  for 
the  disinfection  of  excreta.  The  organism  lives  for  several  weeks 


SPIRILLACE.E   AND   THE   DISEASES   CAUSED  BY   THEM        347 

in  surface  waters  but  certain  waters,  as  for  example  the  Ganges 
River,  destroy  the  cholera  spirilla  very  quickly.  This  property- 
has  been  ascribed  to  a  weak  acidity  of  the  water. 


FIG.  138. — Cholera   vibrios,    longer    forms  at  higher  magnification,  showing  long 
flagella.     (From  Kolle  and  Schurmann  after  Zettnow.) 

Animals  are  not  naturally  susceptible  to  cholera.     Koch  gave 
to  a  guinea-pig  5  c.c.  of  a  5  per  cent  solution  of  sodium  carbonate 


FIG.  139. — Involution  forms  of  the  spirillum  of  cholera.     (Van  Ermengen.) 

through  a  tube,  and  then  5  to  10  c.c.  of  water  containing  cholera 
spirilla.     The  animal  then  received  i  c.c.  of  tincture  of  opium 


348  SPECIFIC   MICRO-ORGANISMS 

per  200  grams  of  body  weight,  injected  into  the  peritoneal  cavity. 
In  this  way  .a  condition  resembling  cholera  in  man  was  induced, 
and  the  animal  died  in  24  to  36  hours.  Autopsy  revealed  severe 
enteritis,  and  abundant  cholera  spirilla  in  the  intestine.  Similar 
results  may  be  obtained,  however,  when  other  organisms  are 
substituted  for  the  cholera  germs  in  this  procedure.  Intravenous 
injection  of  cultures  into  rabbits,  and  feeding  of  virulent  cultures 
to  very  young  rabbits  gives  rise  to  rather  typical  cholera  in  many 
of  the  animals.  Intraperitoneal  injection  of  cultures  into  guinea- 
pigs  gives  rise  to  fatal  peritonitis.  Pigeons  are  relatively  immune. 

The  poisons  of  the  cholera  germ  are  intimately  connected 
with  the  substance  of  the  living  cell.  Culture  filtrates  are 
slightly  or  not  at  all  poisonous.  The  dead  bacterial  cells  are 
poisonous,  but  the  poison  in  them  is  a  very  labile  substance  and 
readily  altered  by  heat.  It  seems  to  become  soluble  when  the 
cell  disintegrates,  and  this  may  explain  the  poisonous  properties 
sometimes  observed  in  the  nitrates  of  older  cultures. 

Immunity  to  this  organism  was  obtained  by  Pfeiffer  by  inject- 
ing non-fatal  doses  into  guinea-pigs.  When  a  small  amount  of 
culture  is  injected  into  the  peritoneal  cavity  of  such  an  immune 
animal,  the  bacteria  become  quickly  clumped  together  and  are 
then  rapidly  disintegrated  and  dissolved  in  the  peritoneal  fluid. 
This  is  known  as  Pfeiffer's  phenomenon  and  was  the  first  example 
of  cytolysis  to  be  observed.  The  solution  of  the  bacteria  sets 
free  their  poison  and  if  a  very  large  dose  has  been  injected  the 
animal  may  be  killed  by  this  poison  regardless  of  his  immunity 
to  the  living  germs. 

Asiatic  cholera  seems  to  have  existed  in  India  for  many 
centuries  and  there  are  reliable  records  of  its  occurrence  there 
in  the  sixteenth,  seventeenth  and  eighteenth  centuries.  The 
first  recognized  great  world  invasion  of  cholera  began  in 
1817  and  ended  in  1823.  Succeeding  pandemics  occurred  in 
1826-1837,  1846-1862,  and  1864-1875.  The  fifth  invasion  began 
in  1883  and  ended  shortly  after  the  great  outbreak  at  Hamburg 
in  1892.  The  sixth  epidemic  began  in  1902  and  has  involved 


SPIRILLACE.E   AND    THE   DISEASES    CAUSED   BY    THEM        349 

Egypt,  Russia,  Turkey  and  Italy.  The  fifth  and  sixth  invasions 
have  been  very  much  restricted,  largely  without  doubt  because 
of  the  modern  methods  founded  upon  knowledge  of  its  causation. 
Cholera  was  epidemic  in  the  United  States  in  1833-35,  1848-54, 
1871-73,  and  there  were  a  few  cases  in  1893  and  again  in  1910. 
The  disease  occurs  as  a  protracted  epidemic  in  which  the  infection 
passes  from  person  to  person,  and  as  an  explosive  epidemic  in 
which  many  people  are  stricken  at  once  as  a  result  of  con- 
tamination of  the  public  water-supply. 

The  causal  relationship  of  Spirillum  cholera  to  human  Asiatic 
cholera  is  no  longer  questioned.  Several  laboratory  workers 
among  them  R.  Pfeiffer  and  E.  Oergel,  have  suffered  typical 
attacks  of  the  disease  as  a  result  of  accidental  laboratory  inocula- 
tion. Dr.  Oergel  received  some  peritoneal  fluid  from  an  inocu- 
lated guinea-pig  into  his  mouth  and  he  died  of  cholera.  Petten- 
koffer  and  Emmerich,  in  order  to  disprove  the  supposed  causal 
relation  of  this  organism  to  cholera,  took  some  alkaline  water 
and  then  water  containing  a  minute  quantity  of  a  fresh  culture. 
The  former  investigator  had  a  severe  diarrhea  and  the  latter  a 
severe  and  dangerous  attack  of  typical  cholera  from  which  he 
eventually  recovered.  The  organism  was  recovered  from  the 
stools  in  all  these  instances. 

The  cholera  spirilla  enter  the  body  with  the  food  and  drink 
and  if  they  escape  the  germicidal  action  of  the  gastric  juice  they 
may  establish  themselves  in  the  intestine.  In  an  acute  case  of 
cholera  they  multiply  here  enormously  and  induce  a  severe 
enteritis  in  which  large  quantities  of  fluid  are  secreted  into  the 
lumen  of  the  intestine  and  discharged  from  the  rectum  along  with 
bits  of  desquamated  epithelium  and  enormous  numbers  of  cholera 
spirilla.  The  germs  do  not  pass  through  the  intestinal  wall,  but 
they  multiply  on  and  in  the  intestinal  epithelium  as  well  as  in 
the  intestinal  contents.  The  general  symptoms,  shock,  coma 
and  the  ultimate  death,  seem  to  be  due  in  part  to  the  absorption 
of  poisons  from  the  intestine  and  in  part  to  the  severe  local  irrita- 
tion in  the  abdomen. 


3  so  SPECIFIC  MICRO-ORGANISMS 

The  bacteriological  diagnosis  depends  altogether  upon  the 
recognition  of  the  cholera  germ  in  the  feces.  During  an  epidemic 
of  the  disease  a  probable  diagnosis  in  the  individual  case  may  be 
made  by  mere  microscopic  examination  of  stained  preparations 
of  the  mucous  flakes  in  the  stools.  The  presence  of  abundant 
curved  rods  arranged  parallel  to  each  other  is  sufficient  for  a 
probable  diagnosis.  The  problem  presents  itself  in  a  different 
phase  when  it  is  necessary  to  recognize  the  first  case  of  cholera  in 
a  given  locality.  Here  it  is  necessary  to  follow  up  the  microscopic 
diagnosis  by  cultures  on  gelatin  plates,  agar  plates  and  in  pepton 
solution,  and  the  identification  of  the  cultured  organisms  by 
agglutinating  them  with  a  known  cholera-immune  serum  in  high 
dilution  (i  :iooo).  The  serum  should  be  powerful  enough  in  a 
dilution  of  i:  10,000  to  agglutinate  very  definitely  the  culture 
used  in  producing  it.  The  examination  of  immigrants  for  the 
detection  of  cholera  carriers  also  requires  culture  work.  The 
stool  should  be  passed  naturally,  but  a  dose  of  salts  is  permissible 
if  there  is  too  great  delay.  About  i  gram  of  feces  is  mixed  with 
50  c.c.  of  sterile  pepton  solution1  in  a  flask,  and  this  is  incubated 
at  37°  C.  f°r  six  to  eight  hours.  A  stained  preparation  is  then 
made  from  the  surface  film  of  the  flask.  If  no  curved  rods  are 
found  in  it,  the  specimen  is  probably  negative.  A  loopful  of  the 
surface  film  should  nevertheless  be  transferred  to  a  tube  of  pepton 
solution  which  is  incubated  for  six  hours  and  again  examined 
microscopically.  If  curved  rods  are  found  microscopically  on 
the  surface  film  of  either  the  first  or  second  culture,  the  problem 
of  differentiating  between  the  cholera  vibrio  and  other  similar 
organisms  is  presented.  Plate  cultures  on  gelatin  at  22°  C.  and 
on  agar  at  37°  C.  should  be  made  and  at  the  same  time  the  trans- 
plantation to  fresh  pepton  solution  should  be  continued  at  six- 
hour  intervals.  After  eighteen  hours,  one  examines  the  plates 
for  typical  colonies  and  subjects  these  to  agglutination  tests  with 
specific  serum  of  high  titre.  The  bacteria  from  the  surface  film 
of  the  pepton  solution  are  also  tested  in  the  same  way.  A  rapid 

1  Pepton  10,  NaCl  10,  NaNO3  o.i,  NaCO3  0.2,  distilled  water  1000. 


SPIRILLACE.E  AND   THE   DISEASES   CAUSED  BY  THEM        3$! 

clearing  of  the  microscopic  field  in  the  agglutination  preparations 
warrants  positive  diagnosis.1 

Similar  principles  are  followed  in  attempting  to  find  cholera 
germs  in  drinking  water.  A  solution  of  pep  ton  100  grams,  salt 
100  grams,  potassium  nitrate  i  gram  and  sodium  carbonate  2 
grams  in  distilled  water  1000  c.c.  is  prepared,  filtered,  distributed 
in  10  flasks  each  of  1000  c.c.  capacity,  and  sterilized.  To  each 
flask  containing  100  c.c.  of  this  sterile  solution,  one  adds  about 
900  c.c.  of  the  suspected  water  and  incubates  the  mixture  at  37°  C. 
for  six  to  eight  hours.  Subcultures  and  microscopic  preparations 
are  made  from  the  surface  films  and  any  curved  bacteria  observed 
are  tested  as  described  above. 

The  prophylaxis  of  cholera  no  longer  rests  upon  the  enforce- 
ment of  quarantine  regulations,  for  it  is  now  known  that  conval- 
escents may  carry  the  vibrio  alive  in  their  intestines  for  many 
weeks.  The  exclusion  of  the  disease  depends  upon  the  bacterio- 
logical examination  of  every  person  coming  from  infected  regions 
before  he  is  allowed  to  land  at  his  destination.  A  water-supply 
system  well  protected  from  fecal  pollution  is  an  element  of  safety 
for  any  community.  The  Hamburg  epidemic  of  1892  illustrated 
this  point.  The  unfiltered  water  taken  from  the  Elbe  near  the 
harbor  carried  the  infection  and  distributed  it  throughout  the  city 
of  Hamburg.  In  the  presence  of  an  epidemic  the  best  protection 
against  contact  infection  is  provided  by  immunization. 

Ferran  in  1884  first  induced  immunity  to  cholera  in  animals 
and  in  man  by  the  subcutaneous  injection  of  living  cultures. 
Haffkine  improved  the  method  so  as  to  make  it  reliable.  He 
employed  a  first  vaccine  of  attenuated  virus  and  a  second  vaccine 
of  high  virulence  with  an  internal  of  five  days  between  the  injec- 
tions. Kolle  introduced  the  use  of  killed  cultures,  employing  a 
single  injection  of  2  mg.  of  growth  from  an  agar  culture  suspended 
in  i  c.c.  of  salt  solution  and  killed  by  heating  an  hour  at  58°  C. 
As  a  result  of  this  treatment  the  agglutinins,  bacteriolysins  and 
opsonins  for  the  cholera  vibrio  are  increased.  Practically  such 

1  Krumwiede,  Pratt  and  Grund,  Journ.  Infect.  Diseases,  1912,  Vol.  X,  pp.  134-141. 


352  SPECIFIC   MICRO-ORGANISMS 

vaccination  has  resulted  in  a  reduction  in  case  incidence  to  about 
one-half  and  in  mortality  rate  to  about  one- fourth  that  observed 
among  the  unvaccinated. 

Spirillum  (Vibrio)  Metchnikovi. — This  curved  organism  was 
found  by  Gamaleia  in  1887  in  the  feces  and  in  the  blood  of  chickens 
suffering  from  enteritis.  Morphologically  and  in  cultures  this 
organism  resembles  Sp.  cholera  very  closely.  It  has  a  single 
flagellum.  The  growth  and  liquefaction  of  gelatin  seems  to  be 
somewhat  more  rapid  in  the  case  of  Sp.  metchnikovi,  and  it  usually 
produces  a  larger  amount  of  indol.  Accurate  differentiation  is 
possible  only  by  animal  experimentation  and  by  testing  with 
anti-sera.  A  minute  quantity  of  culture  of  Sp.  metchnikovi  in- 
troduced into  the  skin  of  a  dove  or  chicken  is  sufficient  to  cause 
general  bacteremia  and  death,  whereas  even  large  doses  (4  mg.) 
of  true  cholera  organisms  introduced  into  such  a  skin  wound  are 
without  effect.  Sp.  metchnikovi  is  also  much  more  virulent  for 
guinea-pigs.  Agglutination  and  bacteriolytic  tests  with  specific 
sera  also  differentiate  the  two  organisms. 

Spirillum  (Vibrio)  Finkler -Prior. — Finkler  and  Prior  in  1885 
isolated  this  organism  from  the  feces  in  cholera  nostras.  Morpho- 
logically it  resembles  the  cholera  vibrio  very  closely.  Indol  is 
not  produced.  It  is  apparently  non-pathogenic. 

Spirillum  Tyrogenum  (Vibrio  Deneke). — This  organism  was 
isolated  from  old  cheese.  It  resembles  the  cholera  vibrio  but 
does  not  form  indol  and  appears  not  to  be  pathogenic. 

A  large  number  of  other  cholera-like  organisms  have  been 
isolated  in  the  Various  examinations  for  the  cholera  germ.  Some 
of  these  can  be  differentiated  morphologically,  as  they  possess 
more  than  one  flagellum.  Others  fail  to  produce  indol  or  show 
other  cultural  difference  from  the  true  cholera  organism.  In 
some  instances  differentiation  depends  almost  altogether  upon 
the  agglutination  test.  This  latter  has  come  to  be  regarded  as 
most  important  in  the  accurate  recognition  of  the  cholera  organ- 
ism and  its  differentiation  from  other  vibrios. 


CHAPTER  XXV. 
SPIROCH.ETVE. 

Spirochaeta  Plicatilis. — Ehrenberg  in  1833  observed  this  long 
slender  spiral  organism  in  swamp  water.  It  occurs  commonly 
in  stagnant  water  among  the  algae  which  grow  there  and  has  also 
been  found  in  sea  water.  The  cell  is  about  0.75/4  in  thickness  and 
20  to  500/1  in  length.  It  moves  by  rotation  and  also  by  bending 
of  the  thread.  Multiplication  takes  place  by  transverse  division, 
sometimes  occurring  simultaneously  at  many  points  in  a  filament 
so  that  many  short  forms  result.  This  organism  is  regarded  as 
the  type  species  of  the  genus  Spirochceta. 

A  number  of  saprophytic  spirochetes  are  known.  Dobell1 
has  made  a  careful  study  of  several  species,  not  only  free-living 
but  also  parasitic  spirochetes,  directing  special  attention  to  their 
systematic  relationships.  He  concludes  that  the  spirochetes 
belong  to  the  bacteria  and  that  they  agree  with  the  bacteria  in 
their  structure  in  all  respects  except  the  organs  of  locomotion. 
Concerning  the  flagella  he  seems  to  be  doubtful. 

Spirochaeta  Recurrentis. — Obermeier  in  1873  described  the 
slender  spiral  organism  first  seen  by  him  in  1868  in  the  blood  in 
cases  of  relapsing  fever.  Ross  and  Milne  observed  a  similar 
organism  in  man  in  Uganda  in  1904  and  Button  and  Todd  in  the 
same  year  demonstrated  the  presence  of  a  spirochete  in  the  blood 
in  the  African  tick  fever  of  the  Congo.  In  1905  a  similar  organ- 
ism was  found  in  a  case  of  relapsing  fever  in  New  York  City. 
The  disease  has  also  been  recognized  in  Russia  and  in  India. 
The  spirochetes  have  been  successfully  inoculated  into  monkeys 
and  into  rats,  and  various  strains  from  different  parts  of  the 
world  have  thus  been  made  available  for  comparative  study  in 

1  Archiv.f.  Protistenkunde,  1912,  Bd.  XXVI,  pp.  117-240. 
23  353 


354  SPECIFIC  MICRO-ORGANISMS 

the  same  laboratory.  There  are  certain  differences  between 
these  spirochetes  of  human  relapsing  fever,  and  several  distinct 
varieties  (or  species?)  are  recognized.  We  shall  consider  them 
as  varieties  of  Sp.  recurrentis. 

Spirochaeta  Recurrentis  var.  Duttoni. — This  is  the  spirochete 
of  Congo  tick  fever  discovered  by  Button  and  Todd  in  1904.  It 
is  about  0.45 /z  in  thickness  and  24  to  30 ju  in  length.  The  organism 
has  been  cultivated  by  Noguchi1  in  ascitic  fluid  containing  sterile 
tissue  and  covered  by  paraffin  oil.  The  African  tick  fever  caused 


FIG.  140. — Spirochaetae  of  relapsing  fever  in  blood  of  a  man.     (After  Kolle  and 

Wassermann.) 

by  this  organism  is  one  of  the  most  fatal  of  the  relapsing  fevers. 
The  tick  remains  infective  for  a  very  long  time  and  also  transmits 
the  infection  to  its  offspring  through  the  egg.  Other  insects,2 
fleas  and  lice,  are  also  capable  of  transmitting  the  infection. 

Spirochaeta  Recurrentis  var.  Rossii  (Kochi). — This  organism 
occurs  in  the  blood  of  relapsing  fever  of  East  Africa.  It  resembles 
Sp.  duttoni  very  closely.  Noguchi  obtained  cultures  readily  in 
ascitic  fluid  containing  sterile  tissue. 

Spirochaeta  Recurrentis  var.  Novyi.3 — This  organism  is 
more  slender  than  the  two  preceding  varieties,  measuring  about 

1  Journ.  Exp.  Med.,  1912,  Vol.  XVI,  pp.  199-210. 

2  Nuttall,  Johns  Hopkins  Hosp.  Bull.,  1913,  Vol.  XXIV,  pp.  33-39. 

3  Novy  and  Knapp:  Journ.  Inf.  Diseases,  1906,  Vol.  Ill,  pp.  291-393. 


355 

0.31  in  thickness.  The  relapsing  fever  in  which  it  occurs  has 
been  observed  in  South  America.  Noguchi  has  obtained  cul- 
tures by  the  same  methods  as  he  employed  for  Sp.  rossii,  but  the 
cultivation  is  more  difficult. 

Several  other  varieties  of  spirochetes,  which  cause  relapsing 
fever  in  man,  have  been  recognized.  The  spirochete  concerned 
in  any  case  seems  to  be  able  to  infect  several  species  of  insects  and 


^^•••••••P^^'— 

FIG.  141. — Spirochczta  recurrentis  (novyi).     Organisms  of  different  lengths  in  the 
blood  of  a  white  rat.     X  1500.     (After  Novy  and  Knapp.) 

to  be  transmitted  to  a  new  mammalian  host  by  them.  Further- 
more one  species  of  insect  seems  to  be  capable  of  transmitting 
any  one  of  these  spirochetes.1 

The  diagnosis  of  relapsing  fever  depends  upon  recognizing  the 
characteristic  spirochetes  in  the  blood  during  the  febrile  attack. 
Their  recognition  offers  little  difficulty,  as  a  rule,  but  they  may  be 
overlooked  by  a  beginner.  In  doubtful  cases  it  is  well  to  search 

1  Nuttall:  Johns  Hopkins  Bull,  1913,  Vol.  XXIV,  pp.  33-39. 


356  SPECIFIC   MICRO-ORGANISMS 

the  fresh  drop  of  blood  not  only  by  direct  central  illumination 
with  a  yellow  light  but  also  by  means  of  dark-field  illumination, 
and  to  examine  thin  films  made  by  mixing  India  ink  3  parts  with 
the  blood  i  part  and  spreading  very  thin.  Finally  thin  blood 
films  should  be  stained  and  examined.  The  inoculation  of  white 
rats  with  i  to  5  c.c.  of  blood  conveys  the  infection  to  them  and 
the  parasites  appear  in  the  blood  of  the  animal  2  to  4  days  after 
inoculation.  The  spirochetes  may  vanish  from  the  blood  with 
marvelous  rapidity. 

Spirochaeta  Anserina. — Sacharoff  in  1890  discovered  this 
spiral  organism  in  the  blood  of  geese  suffering  from  a  serious 
disease  in  the  Caucasus.  Ducks  and  chickens  are  also  susceptible. 
The  spirochete  is  about  o.5/z  thick  by  10  to  20/1  long.  It  is  con- 
sidered by  Nuttall  to  be  indentical  with  the  Sp.  gallinarum  of 
Marchoux  and  Salimbeni. 

Spirochaeta  Gallinarum. — Marchoux  and  Salimbeni  in  1903 
discovered  this  organism  in  the  blood  of  diseased  chickens  at 
Rio  Janeiro.  The  organism  is  0.5/4  thick  and  15  to  20^  long. 
The  disease  is  transmitted  by  means  of  the  fowl  tick  Argas  minia- 
tus  (persicus?},  most  effectively  when  the  tick  is  kept  at  a  tempera- 
ture of  30°  to  35°  C.  In  cold  climates  the  disease  is  unknown. 
Leishman  and  Hindle  have  studied  very  carefully  the  changes 
which  the  spirochetes  pass  through  in  the  body  of  the  insect. 
They  found  numerous  exceedingly  minute  "coccoid  bodies"  in 
the  cells  of  the  Malpighian  tubules.  These  minute  bodies  are 
considered1  to  be  the  products  of  a  fragmentation  of  spirochetes 
and  to  be  capable  of  again  growing  into  typical  spirochetes.  If 
the  view  is  correct  these  bodies  necessarily  play  an  important  part 
in  the  infection  of  the  vertebrate  host  and  in  the  inheritance  of 
the  infection  in  the  insect  species. 

Spirochaeta  Muris. — This  is  a  very  short  spirochete  which 
occurs  naturally  in  a  non-fatal  relapsing  fever  of  rats  and  mice. 
It  possesses  one  or  sometimes  two  flagella  on  each  end  and  multi- 
plies by  simple  transverse  fission. 

1  Nuttall:  Harvey  lecture,  1913. 


357 


Spirochaeta  Pallida  (Treponema  Pallidum).  —  Schaudinn  and 
Hoffmann  in  1905  observed  this  slender  spiral  organism  in  pri- 
mary syphilitic  lesions,  in  fluid  obtained  from  swollen  lymph 
glands  in  syphilis  and  in  the  liver  and  spleen  of  a  still-born  syphi- 
litic fetus.  The  occurrence  of  the 
organism  in  syphilitic  lesions  was 
quickly  and  abundantly  confirmed 
by  other  workers.  Cultures  were 
first  obtained  in  collodion  sacs  by 
Levaditi  and  Mclntosh  in  1907. 
Schereschewsky,  and  Muhlens  and 
Hoffmann  obtained  cultures  in  gela- 
tinized horse  serum.  Noguchi1  has 
carried  out  the  most  successful  cul- 
tural work  and  has  succeeded  for 
the  first  time  in  causing  syphilitic 
lesions  in  animals  by  the  inocula- 
tion of  pure  cultures. 

Sp.  pallida  occurs  naturally  only 
in  human  syphilis.  It  is  a  slender 
spiral  0.2  to  0.3  5  n  in  thickness  and 
3.5  to  1  5.  5  /z  in  length.  Its  curves 
are  narrow  and  very  regular.  It  is 

!  ,.,  „  ,,  .  FIG.     142.  —  Film    preparation 

actively  motile,  as  are  all  the  spiro-   from  a  genital  syphilitic  papule;  in 
chetes,  and  has  a  very  slender  fla-    £e.  ce.nter  arf7^wo  specimens  of 

•;  Sptrochata  pallida,  the  other  three 

gellum  at  each  end.      The  Usual  mo-    are  specimens  of  Spirochala  refrin- 

tion  is  that  of  rapid  rotation  on  the  Schaudinn  and 


longitudinal  axis  with  progression, 

but  at  times  there  is  gross  bending  of  the  filament,  especially 
when  the  organism  is  living  under  unfavorable  conditions. 
The  mode  of  division  is  a  somewhat  vexed  question  as  it 
is  in  regard  to  the  whole  group  of  spirochetes.  Transverse 
and  longitudinal  division  have  been  described.  Probably 
the  weight  of  authority1  now  favors  transverse  division  as 

1  Journ.  Exp.  Med.,  1911,  Vol.  XIV,  p.  99;  1912,  Vol.  XV,  p.  90. 


358  SPECIFIC  MICRO-ORGANISMS 

the  sole  mode  of  multiplication,  although  able  adherents  to  the 
opposite  view  are  not  lacking.  The  refractive  index  of  the 
filament  is  not  very  much  greater  than  that  of  serum,  so  that  the 
unstained  organism  is  difficult  to  see  by  direct  illumination. 
Dark-field  illumination  is  more  satisfactory.  Sp.  pallida  in  film 
preparations  stains  with  difficulty  by  ordinary  methods.  Schau- 
dinn  employed  Giemsa's  modification  of  the  Romanowsky  stain. 
Good  results  are  obtained  by  staining  with  solutions  of  the  Roman- 
owsky staining  principles  in  methyl  alcohol  provided  an  excess  of 
methylene-violet  be  present  (see  p.  43).  Tunnincliff2  recom- 
mends staining  with  a  mixture  of  saturated  alcoholic  solution  of 
gentian  violet,  i  part,  in  5  per  cent  carbolic  acid,  9  parts.  Thin 
films  are  essential  but  the  staining  process  requires  only  a  few 
seconds.  In  pieces  of  tissue  the  spirochete  is  best  stained  by  the 
method  of  Leviditi.  For  this  purpose  thin  (i  mm.)  pieces  of 
syphilitic  tissue  are  fixed  in  formalin  (10  per  cent)  for  24  hours  or 
longer  and  hardened  in  95  per  cent  alcohol  for  a  day.  The  alcohol 
is  then  removed  by  soaking  in  distilled  water  and  the  tissue  is  trans- 
ferred to  a  fresh  i  to  3  per  cent  solution  of  silver  nitrate  in  distilled 
water.  This  is  placed  at  37°  C.  in  the  dark  for  three  to  five  days. 
The  tissue  is  next  washed  in  distilled  water  and  placed  in  a  re- 
ducing fluid,  consisting  of  pyrogallic  acid  3  grams,  formalin  (40 
per  cent  formaldehyde)  5  c.c.  and  distilled  water  100  c.c.,  for  one 
to  two  days.  It  is  then  washed  in  distilled  water,  dehydrated, 
embedded  in  paraffin  and  sectioned.  The  spirochetes  are  stained 
a  dense  black  by  this  method.  The  sections  may  be  stained  to 
show  histological  structure  also,  by  applying  methylene  blue  or 
toluidin  blue  to  them  after  they  have  been  fixed  on  the  slide. 

Cultivation  of  Sp.  pallida  has  been  most  successfully  practised 
by  Noguchi.3  He  has  grown  the  organism  in  a  mixture  of  serum 
and  water,  to  which  naturally  sterile  tissue  was  added,  and  in 
ascitic-fluid  agar  with  similar  bits  of  tissue,  always  under  strict 

1  Hoffmann:  Centrabl.  f.  Bakt.,  I  Abt.,  Orig.,  1912,  Bd.  LXVI,  S.  520-523. 

*Journ.  A.M.  A.,  1912,  Vol.  LVIII,  p.  1682. 

3Journ.  Exp.  Med.,  1911,  Vol.  XIV,  p.  99;  1912,  Vol.  XV,  p.  90 


SPIROCH^ET^E 


359 


anaerobic  conditions.  The  technic  of  culture  is  somewhat  diffi- 
cult and  the  original  papers  should  be  consulted  in  detail.  Inocu- 
lation of  the  cultures  into  rabbits  and  monkeys  has  caused  typical 
syphilitic  lesions. 


FIG.  143. — Spirochceta  pallida  stained  by  Levaditi  method.  The  section  shows 
an  infarcted  lymph  vessel  at  the  junction  of  two  branches.  The  lumen  is  filled  with 
leukocytes.  The  spirochetes  follow  the  lymph  vessel  for  the  most  part,  but  are  also 
penetrating  into  the  surrounding  tissue.  (From  Doflein  after  Ehrmann.} 


Noguchi's  luetin  is  prepared  by  grinding  the  solid  medium 
rich  in  spirochetes  in  a  mortar  and  emulsifying  it  in  a  small 
amount  of  fluid.  This  is  then  heated  to  60°  C.  for  an  hour  and 


360  SPECIFIC   MICRO-ORGANISMS 

preserved  by  the  addition  of  0.5  per  cent  carbolic  acid.  The 
final  preparation  contains  many  dead  unbroken  spirochetes. 

Syphilis  is  an  inoculation  disease  which  has  been  widely 
prevalent  throughout  the  civilized  world  since  the  early  part  of 
the  1 6th  century.  Transmission  takes  place  by  direct  contact 
and  in  the  great  majority  of  instances  by  venereal  contact,  although 
many  authentic  cases  of  transmission  by  means  of  intermediate 
objects  are  known.  The  spirochete  is  able  to  live  for  some  hours 
outside  the  body  if  drying  is  prevented.  The  primary  lesion 
develops  at  the  point  of  inoculation  about  two  weeks  after  that 
event,  first  as  a  papule,  which  becomes  vesicular  and  ulcerates, 
remaining  indolent  for  several  weeks.  The  neighboring  lymph 
glands  become  swollen.  The  secondary  manifestations  occur 
about  a  month  later  as  a  general  macular  or  sometimes  papular 
eruption  on  the  skin,  together  with  sore  throat  and  ulcerated 
patches  in  mouth.  The  skin  eruption  does  not  itch.  Subsequent 
to  this  stage  there  may  be  local  necrotic  lesions  (gummata)  in 
various  parts  of  the  body,  or  low-grade  inflammatory  changes  in 
the  meninges  and  central  nervous  system.  Bacteriological 
methods  of  diagnosis  are  of  assistance  in  some  cases  in  all  the 
various  stages  of  syphilis.  Early  in  the  disease  the  spirochetes 
are  relatively  numerous,  in  certain  locations  at  any  rate,  while 
later  the  parasites  may  be  so  few  as  to  render  their  detection 
practically  hopeless  for  diagnostic  purposes.  In  these  later 
stages,  however,  the  presence  of  specific  and  other  antibodies  in 
the  body  fluids  of  the  patient  may  often  be  recognized  and  this 
recognition  employed  as  an  aid  in  diagnosis. 

Microscopic  examination  of  a  primary  ulcer  is  best  done  by 
means  of  the  dark-field  illumination.  For  this  purpose  the  ulcer 
(which  should  not  have  been  treated  with  mercurials)  is  carefully 
cleansed  and  a  few  drops  of  freshly  exuded  serum  collected  in  a 
glass  capillary,  and  the  usual  slide-cover-glass  preparation  is  made 
with  this  fluid.  Permanent  preparations  are  made  most  easily 
by  mixing  such  serum  with  India  ink  on  a  slide  and  spreading 
the  mixture  in  a  very  thin  layer.  Collargol,  one  part  in  nineteen 


•  361 

parts  of  water,  gives  even  more  satisfactory  preparations1  than 
India  ink.  It  is  used  in  the  same  way.  Thin  films  of  the  serum  - 
on  slides  or  cover-glasses  may  be  stained  as  directed  above.  Micro- 
scopic examination  of  fluid  obtained  by  gland  puncture  or  from 
secondary  lesions  on  the  skin  or  mucous  membranes  is  carried 
out  in  the  same  way.  Serious  confusion  in  the  recognition  of  the 
spirochete  is  likely  to  arise  in  the  case  of  lesions  in  the  mouth  or 
pharynx,  inasmuch  as  some  of  the  normal  mouth  spirochetes  are 
very  similar  in  form  to  Sp.  pallida.  The  presence  of  typical 
spirochetes  in  the  juice  aspirated  from  a  lymph  gland  is  practically 
diagnostic,  and  the  recognition  of  typical  organisms  in  genital 
chancres  or  lesions  on  the  skin  has  considerable  diagnostic  value. 

Inoculation  of  animals  is  of  little  practical  use  in  diagnosis, 
but  it  has  been  possible  by  this  method  to  demonstrate  the  fre- 
quent presence  of  Sp.  pallida  in  the  circulating  blood  in  cases  of 
untreated  secondary  syphilis. 

The  detection  of  antibodies  in  the  blood  of  the  patient  is  under- 
taken in  two  ways,  first  by  the  complement-fixation  ( Wassermann) 
test  and  second  by  the  luetin  test.  For  the  complement-fixation2 
test,  as  performed  at  the  Laboratories  of  the  New  York  Post- 
Graduate  Medical  School  and  Hospital  by  Dr.  R.  M.  Taylor,3 
the  following  are  employed: 

1.  The  red  blood  cells  are  obtained  by  defibrinating  fresh 
sheep's  blood,  filtering  it  through  paper  if  necessary  to  remove 
fragments  of  clot,  separating  the  cells  in  the  centrifuge  and  wash- 
ing them  four  times  with  0.9  per  cent  salt  solution.     Finally  i  c.c. 
of  the  corpuscles  as  packed  by  the  centrifuge  is  suspended  in 
19  c.c.  of  0.9  per  cent  salt  solution;  0.2  c.c.  of  this  suspension  is 
arbitrarily  taken  as  the  unit  of  red  blood  cells. 

2.  The  complement  is  obtained  by  drawing  5  to  10  c.c.  of 
blood  from  a  large  guinea-pig  by  cardiac  puncture.     This  blood 
is  transferred  to  a  Petri  dish,  allowed  to  clot,  incubated  at  37°  C. 

1  Harrison:  Journ.  Roy.  Army  Med.  Corps,  1912,  Vol.  XIX,  p.  749. 

2  For  a  detailed  discussion  see  Citron-Garbat,  Immunity,  Phila.,  1912;  Simon, 
Infection  and  Immunity,  Phila.,  1912. 

3 1  am  indebted  to  Dr.  Taylor  for  the  details  of  this  procedure. 


362  SPECIFIC  MICRO-ORGANISMS 

for  30  minutes  and  then  refrigerated.  The  separated  serum  is 
then  drawn  off  with  a  pipette  and  2  c.c.  of  it  are  mixed  with  18  c.c. 
of  cold  0.9  per  cent  salt  solution.  This  10  per  cent  solution  of 
guinea-pig's  serum  is  kept  in  a  cold  place,  preferably  immersed  in 
ice  water.  It  is  prepared  on  the  day  it  is  to  be  used.  The  unit 
of  complement  is  contained  in  0.2  c.c.  of  this  solution. 

3.  The  hemolytic  amboceptor  is  prepared  by  injecting  2  c.c. 
of  thoroughly  washed  (five  times)  sheep's  corpuscles  intravenously 
into  a  large  rabbit  at  intervals  of  three  days,  until  four  injections 
have  been  given.  Ten  days  after  the  last  injection  the  animal  is 
allowed  to  fast  for  12  hours  and  the  blood  is  then  aseptically 
drawn  from  the  carotid  artery,  allowed  to  clot  and  the  serum 
separated  by  standing  at  37°  C.  for  two  to  five  hours.  The  clear 
serum  is  transferred  to  small  glass  ampoules  in  amounts  of  0.5  to 
i.o  c.c.  and  hermetically  sealed.  These  are  then  heated  at  56°  C. 
for  30  minutes  and  stored  in  the  refrigerator.  The  hemolytic 
power  of  this  serum  is  ascertained  by  titration.  The  unit  is  that 
amount  which,  when  mixed  with  0.2  c.c.  (i  unit)  of  corpuscles 
and  0.2  c.c.  (i  unit)  of  complement  and  sufficient  salt  solution 
(0.9  per  cent)  to  make  a  total  volume  of  i  c.c.,  will  cause  complete 
laking  of  the  red  blood  cells  in  exactly  i  hour  after  being  placed 
in  the  incubator  (air)  at  37°  C.  The  unit  of  amboceptor  is  ordi- 
narily contained  in  o.i  c.c.  of  a  dilution  of  i  part  of  serum  in 
1000  to  2000  parts  of  salt  solution.  After  the  strength  is  ascer- 
tained the  diluted  amboceptor  is  made  up  so  that  o.i  c.c.  contains 
i  unit. 

The  amboceptor  is  quite  permanent  under  ordinary  refrigera- 
tor conditions,  but  when  diluted  it  may  deteriorate  after  a  few 
days.  The  relation  of  complement,  red  blood  cells  and  ambo- 
ceptor is  tested  always  immediately  before  undertaking  a  comple- 
ment-fixation test.  If  the  mixture  of  one  unit  of  each  of  these  in 
a  total  volume  of  i  c.c.  produces  complete  hemolysis  at  the  end 
of  an  hour,  the  hemolytic  system  is  considered  satisfactory.  If 
there  is  only  a  slight  discrepancy  this  may  be  corrected  by  em- 
ploying a  little  more  or  a  little  less  (within  limits  of  20  per  cent) 


SPIROCILETJE  363 

amboceptor,  that  is,  down  to  o.c8  c.c.  or  up  to  0.12  c.c.  as  may  be 
necessary  in  place  of  the  usual  o.io  c.c.  If  the  discrepancy  4s 
greater  than  this  it  is  well  to  obtain  a  new  sample  of  complement 
or  of  sheep's  cells  or  of  both.  The  hemolytic  system  should  be- 
have much  the  same  from  day  to  day  when  the  technic  is 
accurate. 

4.  The  patient's  serum  is  obtained  from  5  to  10  c.c.  of  blood 
drawn  from  the  elbow  vein.     The  serum  must  be  free  from  sus- 
pended matter,  centrifugalized  if  necessary.     The  serum  is  heated 
at  54°  to  56°  C.  for  30  minutes  just  before  use. 

5.  The  antigen  is  a  3  per  cent  solution  in  methyl  alcohol  of  the 
acetone-insoluble  lipoids  extracted  by  alcohol  and  ether  from  the 
heart  muscle  of  beef.     The  strength  of  antigen  to  be  used  must  be 
ascertained  by  careful  titration.     A  dilution  of  i  c.c.  of  the  antigen 
in  9  c.c.  of  salt  solution  is  first  prepared.     Then  various  quanti- 
ties, o.i  c.c.,  0.2  c.c.,  0.3  c.c.,  0.4  c.c.  and  0.5  c.c.  of  this  suspension 
are  placed  in  separate  tubes.     To  each  tube  is  added  i  unit  of 
complement  and  sufficient  salt  solution  to  bring  the  total  volume 
to  i  c.c.     The  tubes  are  incubated  i  hour  at  37°  C.  (air).     Then 
one  unit  of  corpuscles  (0.2  c.c.)  and  two  units  of  hemolytic  ambo- 
ceptor (0.2  c.c.)  are  added  and  the  tubes  are  again  incubated  an 
hour.     Of  those  tubes  in  which  hemolysis  is  not  complete,  the  one 
containing   the  least  antigen  marks  the  concentration  at  which 
the  antigen  is  distinctly  anti-complementary.     The  second  test 
of  the  ant'gen  is  now  undertaken.     Various  amounts  of  a  i  to  100 
dilution,  o.oi  c.c.,  0.03  c.c.,  0.05  c.c.,  o.i  c.c.  and  0.2  c.c.,  are  meas- 
ured into  tubes.     To  each  tube  is  then  added  i  unit  of  comple- 
ment, 0.02  c.c.  of  serum  from  an  active  untreated  case  of  syphilis 
and  sufficient  salt  solution  to  make  a  total  volume  of  0.6  c.c. 
The  tubes  are  incubated  an  hour.     Then  i  unit  of  corpuscles 
(0.2  c.c.)  and  2  units  of  hemolytic  amboceptor  (0.2  c.c.)  are  added 
and  the  tubes  are  again  incubated  one  hour.     Of  the  tubes  show- 
ing no  hemolysis  (complete  fixation),  that  one  which  contains  the 
least  antigen  marks  the  lowest  effective  concentration  of  the  an- 
tigen.    This  amount  of  antigen  should  be  very  much  less  than  the 


364  SPECIFIC   MICRO-ORGANISMS 

anti-complementary  amount  ascertained  in  the  first  test.  Ordi- 
narily it  is  about  T-J~g-  of  this  amount.  The  unit  of  antigen  to 
be  employed  should  be  chosen  so  that  it  is  several  times  greater 
than  the  least  effective  quantity  but  still  not  more  than  one-fifth 
to  one-half  the  least  anti-complementary  amount.  Having  chosen 
the  tentative  antigen  unit,  a  third  test  is  applied.  One,  two  and 
four  units  of  antigen  are  placed  in  tubes  and  a  unit  of  corpuscles 
is  added  to  each,  together  with  sufficient  salt  solution  to  make  the 
total  volume  i  c.c.,  and  these  are  incubated  for  an  hour.  The 
corpuscles  should  not  be  laked.  If  they  are  laked  the  antigen  is 
itself  markedly  hemolytic.  A  satisfactory  antigen  should  per- 
form its  specific  function  of  fixing  complement  in  the  presence 
of  a  syphilitic  serum  in  an  amount  which  is  at  most  -g-V  of  the 
amount  which  is  in  itself  either  anti-complementary  or  hemo- 
lytic. It  keeps  well  in  the  refrigerator  as  the  alcoholic  solution. 
The  dilution  for  use  should  be  freshly  prepared. 

The  antigen  is  the  element  in  the  test  which  is  designed  to 
enter  into  chemical  reaction  with  the  specific  substance  in  the 
patient's  blood,  which  is  present  there  as  a  result  of  active  syphi- 
lis. During  the  course  of  this  reaction,  complement  is  absorbed 
or  destroyed.  The  nature  of  the  lipoidophilic  substance1  is  un- 
known. It  behaves  in  the  test  very  much  as  a  specific  immune 
body  would  be  expected  to  behave.  Experience  has  shown  that 
an  antibody  of  this  nature  is  rarely  present  in  other  conditions 
than  active  syphilis  and  that  it  is  present  in  this  disease.  Upon 
the  results  of  this  experience  we  have  to  rely  in  ascribing  diagnos- 
tic value  to  the  test. 

In  performing  a  test  for  diagnosis,  sera  from  several  patients 
should  be  tested  at  the  same  time,  and  one,  two  or  three  sera,  pre- 
viously tested  and  found  to  fix  complement  in  varying  degrees, 
and  at  least  one  serum  known  to  give  a  negative  result,  should 
be  tested  along  with  the  new  samples.  Four  tubes  are  used  for 
each  serum  to  be  tested. 

1  Simon:  Infection  and  Immunity,  Phila.,  1912,  p.  272. 


365 


Tube  No.  i,  back  row 


Tube  No.  2,  back  row 


Complement  i  unit  (o.  2  c.c.) 
Patient's  serum  0.08  c.c. 
Salt  solution  0.32  c.c. 


Complement  i  unit  (0.2  c.c.) 
Patient's  serum  o.oi  c.c. 
Sheep's  corpuscles,  i  unit  (o.  2  c.c.) 
Salt  solution  0.59  c.c. 


Mix  thoroughly  and  incubate  at  37°  C.     i  hour.     Then  add: 


Sheep's  corpuscles  i  unit 

(0.2C.C.). 

Hemolytic  amboceptor  2  units 

(0.2  c.c.). 

0.4  c.c.1 

Nothing. 

Mix  thoroughly  and  incubate  for  i  hour,  recording  the  progress  of  hemolysis  at. 
intervals  of  15  minutes.     Then  refrigerate  16  hours  and  record  the  final    reading. 


Tube  No.  3,  front  row 


Tube  No.  4,  front  row 


Complement  i  unit  (o.  2  c.c.) 
Patient's  serum  0.02  c.c. 
Antigen  i  unit  (o.  i  c.c.) 
Salt  solution  o.  28  c.c. 


Complement  i  unit  (0.2  c.c.) 
Patient's  serum  o.  04  c.c. 
Antigen  i  unit  (o.  i  c.c.) 
Salt  solution  o.  26  c.c. 


Mix  thoroughly  and  incubate  at  37°  C.     i  hour.   Then  add: 


Sheep's  corpuscles  i  unit  } 

(0.2C.C.).  'o/LCC1 

Hemolytic  amboceptor  2  units   ( 
(0.2  c.c.). 


unit 


Sheep's  corpuscles  i 

(0.2  c.c.).  I  , 

Hemolytic  amboceptor  2  units  [    ' 

(0.2C.C.). 


Mix  thoroughly  and  incubate  for  i  hour,  recording  the  progress  of  hemolysis  at 
intervals  of  15  minutes.     Then  refrigerate  16  hours  and  record  the  final  reading. 


1  The  suspension  of  sheep's  corpuscles  containing  i  unit  in  o.  2  c.c.  and  the  solu- 
tion of  hemolytic  amboceptor  containing  2  units  in  0.2  c.c.  are  quickly  mixed  to- 
gether in  equal  parts,  and  o .  4  c.c.  of  this  homogeneous  mixture  is  added  at  this  point. 
This  procedure  results  in  a  saving  of  time  as  well  as  greater  accuracy. 


366  SPECIFIC  MICRO-ORGANISMS 

Tube  No.  i  should  show  complete  hemolysis  early  in  the  second 
incubation.  Tube  No.  2  should  remain  free  from  hemolysis,  or 
show  only  a  very  slight  amount  at  the  end  of  the  second  incu- 
bation. If  these  have  behaved  properly  and  the  tests  on  the 
known  sera  have  resulted  as  they  did  when  previously  tested, 
then  the  behavior  of  Tubes  3  and  4  is  a  measure  of  the  amount  of 
lipoidophilic  substance  in  the  serum  of  the  patient.  One  dis- 
tinguishes about  eight  different  grades  of  reaction,  from  complete 
fixation  (no  trace  of  hemolysis)  to  no  fixation  (complete  hemolysis) . 

The  luetin  test  is  performed  by  injecting  0.05  c.c.  of  luetin 
intracutaneously  in  two  places  on  the  left  arm  and  at  the  same 
time  0.05  c.c.  of  a  control  suspension,  consisting  of  the  medium 
without  any  growth  of  spirochetes,  at  two  points  on  the  right 
arm.  Local  inflammation  on  the  left  arm,  appearing  in  two  to 
ten  days  and  sometimes  resulting  in  the  formation  of  a  pustule, 
is  regarded  as  a  positive  test.  The  test  is  often  negative  in  the 
earlier  stages  of  syphilis.  H 

The  various  diagnostic  tests  for  syphilis  are  now  extensively  em- 
ployed. Microscopic  search  for  the  spirochete  is  of  value  in  the 
untreated  primary  and  secondary  stages.  The  complement-fixa- 
tion test  becomes  positive  a  few  weeks  after  the  appearance  of  the 
primary  lesion  and  is  generally  regarded  as  indicating  an  active 
syphilitic  process.  The  luetin  test  may  be  positive  in  latent  or 
inactive  syphilis  when  the  Wassermann  is  negative.  Further 
experience  with  the  luetin  test  is  necessary  in  order  to  determine 
its  real  significance. 

Spirochaeta  (Treponema)  Refringens. — This  is  a  relatively 
gross  spirochete  which  occurs  in  primary  syphilitic  lesions  along 
with  Sp.  pallida.  It  seems  to  have  no  pathogenic  properties. 
Noguchi1  has  obtained  pure  cultures  of  it  and  found  them  with- 
out pathogenic  properties  for  rabbits  and  monkeys. 

Spirochaeta  (Treponema)  Microdentium.2 — This  is  one  of  the 
common  spirals  of  the  mouth.  It  may  be  confused  with  Sp.  pal- 

1  Journ.  Exp.  Med.,  1902,  Vol.  XV,  p.  466. 

2 Noguchi:  Journ.  Exp.  Med.,  Vol.  XV,  pp.  81-89. 


367 

lida,  which  it  resembles  in  size  and  shape.  Pure  cultures  have 
been  obtained  by  Noguchi.  Other  spirochetes  of  the  mouth 
have  also  been  cultivated  by  this  investigator  and  there  are  prob- 
ably several  species  of  them. 

Spirochaeta  (Bacillus)  Fusiformis  (Vincenti). — In  an  ulcer  a- 
tive  disease  of  the  tonsils  known  as  Vincent's  angina  there  occur 
very  constantly  large  numbers  of  fusiform  rods  0.3  to  o.Sju  in 
thickness  and  3  to  lo/x  long,  associated  with  spiral  filaments  with 
rather  coarse  windings.  Similar  organisms  occur  in  other  ul- 
cerative  conditions  of  the  mouth  and  pharynx  and  rarely  else- 
where in  the  body.  The  relation  of  these  organisms  to  each  other, 
whether  they  are  distinct  species  or  different  forms  of  the  same 
species,  is  still  unsettled.  Their  etiological  relationship  to  the 
disease  is  also  uncertain.  Tunnicliff1  has  observed  spiral  forms 
in  her  pure  cultures  of  Bacillus  fusiformis.  It  seems  probable 
that  the  spirals  seen  in  the  ulcer  are  to  a  large  extent  the  ordi- 
nary mouth  spirochetes,  but  the  fusiform  bacillus  itself  is  evidently 
a  close  relative  of  the  spirochetes,  as  it  requires  similar  conditions 
for  successful  culture  and  is  able  at  times  to  assume  a  distinctly 
spiral  form  in  culture. 

1  Journ.  Inf.  Diseases,  1906,  Vol.  Ill,  p.  148;  Rosenow  and  Tunnicliff:  Journ 
Inf.  Dis.,  1912.  Vol.  X,  pp.  1-6. 


CHAPTER  XXVI. 
THE  FILTERABLE  MICROBES.  * 

The  Virus  of  Foot-and-mouth  Disease. — This  filterable  or- 
ganism occurs  in  the  vesicles  present  in  the  mouth  and  on  the 
feet  of  the  diseased  animals,  and  also  in  the  milk  of  cows  suffering 
from  foot-and-mouth  disease.  The  virus  was  shown  to  be  filter- 
able by  Loffler  and  Frosch  in  1898.  It  is  rendered  inert  by  heat- 
ing to  50°  C.  for  10  minutes.  Animals  are  immune  after  recovery 
from  the  disease.  Cattle  and  swine  are  naturally  susceptible 
and  a  few  cases  of  the  disease  have  occurred  in  man.  Nothing 
definite  is  known  concerning  morphology  or  cultures.  The  in- 
fection seems  to  be  transmitted  with  the  food  as  well  as  by 
inoculation. 

The  Virus  of  Bovine  Pleuro-pneumonia. — This  organism  is 
present  in  the-  affected  lungs  and  in  discharges  from  the  respira- 
tory tract  of  cattle  suffering  from  pleuro-pneumonia.  Nocard 
filtered  the  virus  through  a  Chamberland  "F"  filter  in  1899.  It 
is  rendered  inert  by  heating  at  58°  C.,  but  retains  its  virulence  in 
glycerine  for  weeks  and  resists  freezing.  Cultures  have  been 
obtained  by  the  collodion-sac  method  by  Nocard  and  Roux.  The 
organisms  in  such  cultures  are  extremely  minute  and  variable  in 
form.  Some  of  them  are  spirals  and  others  approximately  spher- 
ical. Immunity  follows  recovery  from  the  disease,  and  has  been 
induced  artificially  by  inoculation  with  cultures  and  also  by  inocu- 
lation with  virulent  exudate  from  the  lung  of  a  dead  animal  into 
the  subcutaneous  tissue  of  the  tail  of  the  animal  to  be  immunized.1 

The  Virus  of  Yellow  Fever. 2 — This  organism  occurs  in  the  blood 
of  man  at  least  during  the  first  two  or  three  days  of  an  attack  of 

1  Kolle  and  Wassermann,  Handbuch,  1912,  Bd.  I,  S.  928. 

2  The  publications  of  Reed,  Carroll  and  their  associates  have  been  issued  as  a 
volume    entitled  Yellow  Fever,  U.  S.  Senate  Document  No.  822,  6ist  Congress, 
3rd  Session,  1911. 

368 


THE  FILTERABLE  MICROBES 


369 


yellow  fever.  It  was  shown  to  be  filterable  by  Reed,  Carroll, 
Lazear  and  Agramonte  in  1901.  It  passes  through  the  Chamber- 
land  "B"  filter.  It  is  rendered  inert  at  55°  C.  in  10  minutes  and 
even  by  standing  at  room  temperature  for  two  days.  Yellow 
fever  is  an  acute  febrile  disease  of  man  usually  accompanied  by 
jaundice  and  sometimes  by  the  vomiting  of  altered  blood  (black 


aft 


FIG.  144. — Aedes  (Stegomyla)  colipus;  female,     a,  Front  tarsal  claw.     (After  Reed 

and  Carroll^) 

vomit).  It  is  frequently  fatal.  Permanent  immunity  follows 
recovery.  The  disease  is  naturally  transmitted  by  a  blood-suck- 
ing mosquito,  (Stegomyia,  Aedes)  calopus,  which  becomes  capable 
of  inoculating  the  disease  about  twelve  days  after  sucking  blood 
containing  the  virus.  The  mosquito  probably  remains  infective 
as  long  as  it  lives,  and  this  insect  thus  becomes  the  essential  reser- 
voir of  the  virus  of  yellow  fever.  Prophylactic  measures  based 
24 


370  SPECIFIC  MICRO-ORGANISMS 

upon  this  deduction  have  been  remarkably  successful  in  the  sup- 
pression of  the  disease. 

Seidelin1  has  described  a  minute  structure  which  occurs  in  the 
blood  cells  and  in  the  blood  plasma  in  yellow  fever,  which  he  has 
called  Paraplasma  flamgenum  and  regards  as  the  pathogenic 
agent.  The  work  lacks  confirmation  by  other  observers  and  the 
evidence  is  not  yet  convincing.  The  earlier  papers  of  Seidelin  have 
been  severely  criticised  by  Agramonte.1 

The  Virus  of  Cattle  Plague  (Rinderpest). — This  organism 
occurs  in  the  blood,  organs  and  excretions  of  cattle  suffering  from 
the  disease.  It  was  shown  to  be  filterable  by  Nicolle  and  Adil- 
Bey  in  1902,  and  is  able  to  pass  through  the  Chamberland  "F" 
filter.  The  virus  resists  drying  for  four  days  and  remains  active 
for  two  or  three  months  when  spread  on  hay  in  a  dark  place. 
It  is  destroyed  by  distilled  water  in  five  days,  by  glycerin  in  eight 
days  and  rendered  avirulent  in  a  few  hours  by  admixture  of  bile. 
The  disease  is  an  acute  febrile  disorder  characterized  by  severe 
inflammation  of  the  mucous  membranes  and  rapid  emaciation. 
It  is  usually  fatal.  Immunity  follows  recovery  and  is  induced 
artificially  by  injecting  the  bile  of  infected  animals  under  the 
skin  of  the  healthy  cattle.  In  this  way  an  active  immunity  is 
acquired  without  an  evident  attack  of  the  disease. 

The  Virus  of  Rabies. — This  organism  exists  in  the  central 
nervous  system,  the  peripheral  nerves,  the  salivary  glands,  the 
saliva  and  less  frequently  in  other  parts  of  the  body  of  persons 
or  animals  suffering  from  lyssa  or  rabies.  The  virus  was  filtered 
by  Remlinger  in  1903.  It  may  also  be  dialyzed  through  collodion 
sacs.3  The  virus  is  rendered  inert  by  drying  for  two  weeks,  and 
by  heating  at  55°  C.  for  30  minutes,  by  admixture  of  bile  in  a  few 
minutes,  and  by  the  gastric  juice  in  5  hours.  It  remains  virulent 
in  glycerine  for  several  months.  Negri  in  1903  described  certain 
bodies  which  seem  to  occur  in  the  central  nervous  system  in- 
variably and  exclusively  in  this  disease.  They  are  especially 

1  Bull.  Yellow  Fever  Bureau,  1912,  Vol.  II,  pp.  123-242. 

1  Medical  Record,  1912,  Vol.  LXXXI,  pp.  604-607. 

2  Poor  and  Steinhardt,  Journ.  Infect.  Dis.,  1913,  Vol.  XII,  pp.  202-205. 


THE  FILTERABLE  MICROBES 


371 


numerous  in  the  ammon's  horn  of  the  brain  in  cases  of  street 
rabies.  Preparations  should  be  made  from  the  gray  matter  of 
the  brain.  A  bit  of  this  tissue  is  carefully  spread  on  a  slide  by 
exerting  moderate  pressure  upon  it  with  a  second  slide  or  a  cover- 
glass  and  at  the  same  time  moving  it  along  the  surface  of  the  first 


FIG.  145. — Section  through  the  cornu  ammonis  of  brain  of  a  rabid  dog;  stained  by 
the  method  of  Lentz.  Five  Negri  bodies  of  different  sizes  are  shown,  enclosed  within 
the  ganglion  cells.  The  smallest  contains  only  three  minute  granules.  (After  Lentz, 
Centralbl.f.  Bakt,  1907,  Abt.  I,  Vol.  XLIV,  p.  378.) 


slide.  The  film  is  fixed  in  pure  methylic  alcohol  and  stained  with 
Giemsa's  solution,  or  it  may  be  stained  directly  without  fixation 
with  Leishman's  stain.  The  Negri  bodies  are  round  and  some- 
what irregular  in  outline  from  i/i  to  27/^1  in  diameter,  and  usually 
inside  the  nerve  cells.  In  the  interior  of  the  larger  bodies,  smaller 


372  SPECIFIC   MICRO-ORGANISMS 

spherical  structures  of  variable  size  and  number  may  be  seen. 
The  exact  nature  of  the  Negri  bodies  is  uncertain.  Some  stu- 
dents of  rabies  regard  them  as  protozoa,  while  others  consider 
them  to  be  products  of  cell  degeneration.  The  evidence  to  de- 
cide the  matter  is  not  yet  at  hand.  They  seem  to  occur  only  in 
rabies  and  to  be  constantly  present  in  this  disease. 

Lyssa  or  rabies1  is  primarily  a  disease  of  dogs  but  it  occurs  in 
other  mammals  as  well,  usually  as  a  result  of  dog  bites.  In  ani- 
mals inoculated  directly  into  the  brain  with  the  most  virulent 
material  (fixed  virus),  the  symptoms  of  rabies  appear  in  4  to  6 
days  and  death  occurs  on  the  seventh  day.  Inoculation  with  the 
saliva  or  nervous  tissue  of  a  mad  dog  (street  virus)  rarely  causes 
symptoms  before  three  weeks  and  the  onset  may  be  delayed  for 
a  year.  In  fact  many  persons  and  animals  bitten  by  rabid  dogs 
may  fail  to  develop  the  disease  at  all.  This  variability  depends 
upon  the  virulence  and  the  amount  of  virus  and  especially  upon 
the  part  of  the  body  into  which  it  is  introduced.  Bites  upon  the 
face  or  hands,  because  of  the  rich  nerve  supply  and  the  lack  of 
protection  by  clothing,  are  especially  dangerous.  After  the  dis- 
ease has  developed  so  as  to  cause  symptoms,  death  is  inevitable 
in  the  present  state  of  our  knowledge. 

Rabies  may  be  diagnosed  in  an  animal  by  observing  the  course 
of  the  disease,  by  autopsy  and  by  inoculation  of  test  animals  and 
observation  of  the  course  of  the  disease  in  them.  If  the  sus- 
pected animal  be  caged,  the  question  of  rabies  may  be  settled  in  a 
few  days,  for,  if  he  is  mad,  the  raging  stage  will  be  quickly  followed 
by  the  characteristic  paralysis  and  death.  If  the  animal  has  been 
killed,  a  careful  autopsy  may  reveal  the  absence  of  food  from  the 
digestive  tract  and  the  presence  there  of  abnormal  ingested  ma- 
terial (grass,  wood  or  stones),  highly  suggestive  of  rabies.  Mi- 
croscopic examination  of  the  central  nervous  system  may  reveal 
the  Negri  bodies,  characteristic  of  the  disease.  For  confirmation 
of  the  diagnosis  a  portion  of  the  brain  or  spinal  cord,  removed  with- 

1  For  a  general  discussion  of  rabies  see  Gumming:  Journ.  A.  M.  A.,  1912,  Vol. 
LVIII,  pp.  1496-1499. 


THE  FILTERABLE  MICROBES  373 

out  contamination,  should  be  injected  into  the  brain  of  guinea- 
pigs  and  rabbits  and  the  effects  observed.  This  last  test  caF 
ried  out  by  an  experienced  observer  is  the  most  trustworthy 
of  all. 

The  Pasteur  treatment  of  rabies  is  designed  to  induce  immu- 
nity after  the  person  has  been  bitten  and  before  the  disease  has  had 
time  to  develop.  Pasteur1  first  demonstrated  the  possibility  of 
this  by  experimental  work  on  dogs,  and  the  subsequent  use  of 
the  method  in  man  has  been  remarkably  successful  and  the  dis- 
ease is  practically  always  prevented  if  the  treatment  is  begun 
directly  after  infliction  of  the  infecting  wound.  The  first  essen- 
tial is  thorough  cauterization  of  the  wound,  best  with  concentrated 
nitric  acid  under  anesthesia.  The  patient  is  then  injected  sub- 
cutaneously  with  emulsions  of  the  spinal  cords  which  have  been 
removed  from  rabbits  dying  of  rabies  after  inoculation  with  the 
fixed  virus,  and  which  have  been  dried  by  hanging  in  bottles 
over  caustic  soda  for  some  time.  The  first  injection  is  prepared 
from  cords  hung  for  14  and  13  days,  the  second  from  cords  hung 
1 2  and  1 1  days,  and  so  on  until  the  three-day  cord  is  reached  on 
the  seventh  or  eighth  day  of  the  treatment.  The  series  from 
five-day  down  to  three-day  cords  is  then  repeated  several  times, 
the  whole  treatment  lasting  about  21  days.  The  course  of  treat- 
ment is  varied  somewhat  according  to  the  urgency  of  the  case  and 
the  severity  of  the  wounds  inflicted.  It  is  most  effectively  carried 
out  at  special  Pasteur  institutes  devoted  to  this  work,  but  the 
material  for  injection  may  be  shipped  for  some  distance  when 
necessary. 

The  Virus  of  Hog  Cholera. — Dorset,  Bolton  and  McBryde, 
continuing  the  investigations  of  de  Schweinitz,  demonstrated  in 
1905  the  presence  of  a  filterable  agent  in  the  blood  of  hogs  suffering 
from  hog  cholera,  capable  of  causing  the  disease  upon  injection 
into  healthy  animals.  It  passes  through  the  Chamberland  "B" 
and  "F"  filters.  It  leaves  the  body  in  the  urine  and  probably 
also  in  other  excretions,  and  seems  to  enter  the  new  victim  with 

1  Vallery-Radot,  The  Life  of  Pasteur,  1911,  Vol.  II,  p.  188. 


374  SPECIFIC  MICRO-ORGANISMS 

the  food  and  drink.  The  virus  resists  drying  for  three  days, 
remains  alive  in  water  for  many  weeks  and  in  glycerine  for  eight 
days.  It  is  destroyed  at  60°  to  70°  C.  in  an  hour. 

King,  Baeslack  and  Hoffman1  have  found  a  short,  rather  thick, 
actively  motile  spirochete,  Spiroch&ta  suis,  in  the  blood  in  forty 
cases  of  hog  cholera,  together  with  abundant  granules  which  may, 
perhaps,  represent  a  stage  of  this  organism.  The  spirochete  has 
not  been  found  in  healthy  hogs.  It  seems  probable  that  this 
organism  may  prove  to  be  the  causative  agent  of  the  disease,  but 
further  evidence  is  necessary  to  demonstrate  this  relationship. 

Hog  cholera  is  an  extremely  contagious  disease  of  hogs,  fre- 
quently fatal,  characterized  by  fever  and  by  ulcerations  in  the 
intestine.  Immunity  follows  recovery  and  is  induced  artificially 
by  the  injection  of  serum  from  a  hyperimmune  hog  (passive 
immunity)  and  by  the  injection  of  such  serum  together  with  viru- 
lent blood  from  a  hog  sick  with  the  disease  (combined  passive 
and  active  immunity) . 

The  Virus  of  Dengue  Fever. — Ashburn  and  Craig  showed  in 
1907  that  the  virus  of  this  disease  exists  in  the  blood  of  the  pa- 
tients and  that  it  is  filterable.  The  disease  is  probably  trans- 
mitted by  the  mosquito  Culex  fatigans.  Apparently  the  analogy 
to  yellow  fever  is  rather  close. 

The  Virus  of  Phlebotomus  Fever. — Doerr  in  1908  demon- 
strated a  filterable  virus  in  the  blood  of  persons  suffering  from 
the  benign  three-day  fever  of  Malta  and  Crete.  The  disease  is 
rather  widely  distributed  in  tropical  countries.  It  is  transmitted 
by  the  sand-fly  Phlebotomus  papatasii. 2 

The  Virus  of  Poliomyelitis. — Several  investigators,  among 
themrFlexner  and  Lewis,  demonstrated  in  1899  the  presence  of  a 
filterable  virus  in  the  central  nervous  system  of  patients  suffering 
from  infantile  paralysis.  The  virus  also  occurs  in  the  nasal  mucus 
and  in  the  blood.  It  survives  in  glycerine  for  a  month,  also  re- 
sists freezing  for  weeks,  and  is  rendered  inert  at  45°  to  50°  C.  in 

1  Journ.  Infect.  Dis.,  1913,  Vol.  XII,  pp.  39-47;  PP-  206-235. 

2Birt.  Journ.  Roy.  Army  Med.  Corps,  1910,  Vol.  XIV,  pp.  236-258. 


THE  FILTERABLE  MICROBES  375 


30  minutes.     It  is  quickly  destroyed  by  hydrogen  peroxide  and^ 
by  menthol. 

Flexner  and  Noguchi1  have  obtained  cultures  of  the  organism 
in  ascitic  fluid  containing  sterile  tissue  and  covered  with  paraf- 
fin oil,  and  in  this  medium  rendered  solid  by  admixture  of  agar. 
The  colonies  are  made  up  of  minute  globose  bodies  0.15  to  0.30/4 
in  diameter.  Similar  bodies  have  been  identified  in  the  nervous 
tissue  from  cases  of  the  disease.  It  seems  probable  that  this 
structure  is  a  living  organism  and  the  microbic  cause  of  poliomye- 
litis, especially  as  inoculation  of  monkeys  with  the  cultures  has 
given  rise  to  the  disease. 

Poliomyelitis  or  infantile  paralysis  occurs  in  epidemics  and 
also  sporadically,  attacking  children  and  young  adults.  It  is 
characterized  by  digestive  disturbance  and  fever,  which  may  be 
very  mild,  followed  by  paralysis  of  one  or  more  extremities  as  a 
rule.  Death  may  occur,  but  recovery  with  permanent  paralysis 
is  the  usual  result.  The  mode  of  transmission  is  unknown. 
Rosenau  is  inclined  to  ascribe  considerable  importance  to  the 
stable  fly,  Stomoxys  calcitrans,  as  the  transmitting  agent.  Other 
modes,  especially  direct  contact,  food,  and  healthy  carriers  also 
need  to  be  considered. 

The  Virus  of  Measles.  —  Goldberger  and*  Anderson2  in  1911 
succeeded  in  inoculating  monkeys  with  measles  and  demonstrated 
the  presence  of  the  virus  in  the  blood  and  in  the  secretions  of  the 
nose  and  mouth,  and  in  filtrates  of  these  fluids.  The  organism 
passes  through  the  Berkefeld  filters.  The  virus  is  destroyed  at 
55°  C.  in  15  minutes. 

The  Virus  of  Typhus  Fever.  —  Nicolle,  Conor  and  Conseil  in 
1910  transmitted  typhus  fever  to  monkeys  by  means  of  serum 
which  had  passed  through  a  Berkefeld  filter.  Ricketts  and  Wilder 
failed  to  obtain  infective  filtrates  in  their  study  of  Mexican  ty- 
phus. Typhus  is  an  acute  febrile  disease,  widely  distributed  but 
not  very  prevalent  in  any  locality.  Apparently  it  is  not  con- 

1  Journ.  A.  M.  A.,  1913,  Vol.  LX,  p.  362. 
2Journ.  A.  M.  A.}  1911,  Vol.  LVII,  pp.  971-972. 


SPECIFIC   MICRO-ORGANISMS 

tagious1  but  is  transmitted  from  man  to  man  by  body  lice  (Pedi- 
culus  vestimenti).  Immunity  follows  recovery. 

The  Virus  of  Small-pox. — The  virus  of  this  disease  was  shown 
to  be  filterable  by  Casagrandi  in  1908.  The  vaccine  virus,  which 
is  generally  considered  to  be  the  same  organism,  had  been  pre- 
viously filtered.  The  organism  passes  through  the  coarser  Cham- 
berland  filters.  The  virus  resists  drying  for  several  weeks  and 
remains  active  in  glycerine  for  eight  months,  but  is  quickly  ren- 
dered inert  by  bile  and  by  sodium  oleate.  It  is  also  destroyed  by 
heating  at  58°  C.  for  15  minutes.  Cell  inclusions,  which  were 
described  by  Guarnieri  in  1892,  are  considered  by  some  to  repre- 
sent forms  of  the  pathogenic  agent. 

Small-pox  is  an  acute  disease  of  man  characterized  by  a  general 
eruption  on  the  skin,  at  first  papular,  then  vesicular  and  pustu- 
lar. It  is  highly  contagious  by  direct  association  and  by  fomites 
and  is  readily  transmitted  by  placing  bits  of  crust  from  dried 
pustules  on  the  nasal  mucous  membrane  or  on  a  scratch  in  the 
skin.  Cow-pox  is  a  milder  disease  which  occurs  naturally  in  cows, 
and  has  also  been  produced  by  inoculating  calves  with  small-pox 
virus.  An  attack  of  either  small-pox  or  cow-pox  is  followed  by 
immunity  to  both  diseases.  Cow-pox  in  man  is  a  comparatively 
mild  disease.  Inoculation  results  in  the  formation  of  a  single 
pustule,  rarely  surrounded  by  secondary  vesicles,  with  slight  illness 
for  a  few  days.  Edward  Jenner  in  1798  discovered  that  cow-pox 
resulting  from  artificial  inoculation  (vaccination)  confers  an  immu- 
nity to  small-pox.  Vaccination  is  now  very  generally  practised 
in  enlightened  communities  and  in  such  places  small-pox  is  practi- 
cally unknown.  The  inoculation  is  best  done  by  making  a  very 
slight  superficial  linear  incision,  about  5  mm.  long,  in  the  epi- 
dermis and  rubbing  into  it  the  vaccine  virus.  The  whole  pro- 
cedure should  result  in  only  a  faint  tinge  of  blood.  When  the 
vesicle  appears  it  should  be  carefully  protected  from  violence. 
A  normal  vaccination  causes  little  inconvenience  and  is  usually 

1  Wilder:  Journ.  Infect.  Dis.,  1911,  Vol.  IX,  p.  9.     Ricketts  and  Wilder :Joiirn. 
A.  M.  A.,  1910,  Vol.  LV,  pp.  309-311. 


THE  FILTERABLE  MICROBES  377 

completely  healed  in  about  4  weeks  after  inoculation.  Failure 
of  the  inoculation  is  not  a  proof  of  immunity.  The  vaccination 
should  be  repeated  until  it  does  take. 

The  Virus  of  Chicken  Sarcoma. — Rous  in  1910  discovered  a 
tumor  in  a  chicken  which  is  histologically  a  typical  spindle-cell 
sarcoma  and  which  he  has  been  able  to  reproduce  in  other  chickens, 
not  only  by  transplantation  but  also  by  inoculation  of  an  agent 
which  can  be  separated  from  the  tumor  cells1  by  nitration  through 
Berkefeld  filters,  as  well  as  by  inoculation  with  tumor  tissue  which 
has  been  dried  and  powdered  and  preserved  in  the  dry  condition 
for  months.  The  filterable  microbe,  or  filterable  agent  as  Rous 
conservatively  calls  it,  is  rendered  inert  by  heating  at  55°  C.  in 
15  minutes,  also  by  the  admixture  of  chicken  bile  or  saponin. 
Two  other  sarcomata  of  the  fowl  have  been  shown  to  be  due  to  a 
filterable  agent  by  the  same  investigator. 

Our  conceptions  of  the  nature  of  filterable  agents  is  at  present 
beginning  to  become  more  definite.  They  are  no  longer  re- 
garded as  necessarily  beyond  the  possibility  of  morphological  study 
and  there  is  good  reason  to  hope  that  the  development  of  improved 
methods  of  study  and  their  careful  application  may  be  able  to 
establish  not  only  the  important  physiological  properties  of  these 
agents  but  their  form  and  perhaps  to  some  extent  their  structure 
as  well.  The  beginning  already  made  is  full  of  promise  for  the 
future.2 

1  Rons  and  Murphy:  Journ.  Exp.  Med.,   1913,   Vol.  XVII,  pp.  219-231.     Pre- 
vious papers  are  cited  there. 

2  A  number  of  other  diseases  have  been  shown  to  be  caused  by  filterable  agents. 
A  brief  mention  of  these  together  with  references  to  the  literature  will  be  found  in 
the  article  by  Wolbach:  Journ.  Med.  Rsch.,  1912,  Vol.  XXVII,  pp.  1-25. 


CHAPTER  XXVII. 


MASTIGOPHORA.1 

Herpetomonas  Muscae  (Domesticae).2  —  This  flagellate  proto- 
zoon  is  commonly  found  in  the  intestine  of  the  house  fly  (Musca 
domestica).  The  cell  body  is  spindle  shaped  (Fig.  146)  and  15  to 
to  2  5  A*  in  length.  The  flagellum  is  of 
about  equal  length  and  contains  two 
stainable  filaments  which  terminate  near 
the  deeply  staining  blepharoplast  situated 
in  the  anterior  part  (flagellated  end)  of 
the  cell.  From  this  blepharoplast  a  deli- 
cate thread  extends  in  the  cytoplasm  to- 
ward the  posterior  end.  The  nucleus  (tro- 
phonucleus)  is  at  the  center  of  the  cell. 
Multiplication  takes  place  by  longitudinal 
division. 

Leptomonas  (Herpetomonas)  Culicis.3 
—  In  the  digestive  tract  of  mosquitoes, 
flagellated  organisms  occur  which  bear  a 

FIG.  146.  —  Herpetomonas 

a,  Normal  indi-  confusing  resemblance  to  trypanosomes. 
Thfy  ™>ltiply  abundantly  in  the  blood 
which  the  insect  ingests  and  are  most 

..      f          .  .       . 

easily  found  in  the  mosquito  near  the  end 
of  digestion  of  a  blood  meal  (48  to  96  hours  after  feeding).  The 
body  is  1  6  to  45  ju  in  length  and  0.5  to  2/4  in  width.  Artificial 
cultures  have  been  obtained  in  the  condensation  water  of  blood- 
agar  and  these  have  been  purified  by  streaking  on  blood-agar 

1  Only  a  few  protozoal  forms  can  be  considered  and  those  very  briefly.     The 
interested  student  should  consult  Doflein:  Protozoenkunde,  III  Auflage,  Jena,  1911. 

2  Prowazek,  Arb.  Kais.  Gesundheitsamt.,  1904,  Bd.  XX,  S.  440. 
3Novy,  MacNeal  and  Torrey,  Journ.  Inf.  Dis.,  1907,  Vol.  IV,  p.  223. 

378 


blepharoplast.        (From 

Doflein  after  Prowazek.) 


MASTIGOPHORA  379 

plates.     The  organism  is  not  known  to  be  capable  of  infecting 
vertebrates. 

Somewhat  similar  flagellates  are  found  in  the  alimentary 
tract  of  various  insects,  where  they  may  be  easily  mistaken  for 
developmental  stages  of  hematozoa.  Trypanosoma  (Herpeto- 
monas)  grayi  which  is  found  in  the  tsetse  fly  Glossina  palpalis 
may  be  mentioned  as  another  example. 


FIG.  147. — Leptomonas  culicis  from  the  digestive  tract  of  a  mosquito.     X   1500. 
(After  Novy,  MacNeal  and  Torrey.) 

Trypanosoma  Rotatorium. — This  organism  is  the  type  species 
of  the  genus  Trypanosoma,  as  this  name  was  first  applied  to  it  by 
Gruby  in  1843.  It  *s  commonly  found  in  small  numbers  in  the 
blood  of  frogs.  The  form  of  the  cell  varies  from  that  of  a  slender 
spindle  to  a  very  broad  and  thick  structure  (Fig.  148).  The 
width  varies  from  5  to  40/1,  and  the  length  from  40  to  8oju.  These 
various  forms  are  probably  stages  in  the  growth  of  the  parasite  but 
it  is  not  impossible  that  they  represent  different  species  parasitic 
in  the  same  animal.  When  the  larger  forms  are  well  stained  the 
typical  structures  of  a  trypanosome  are  distinctly  evident.  The 
large  nucleus  (trophonucleus)  lies  near  the  middle  of  the  body 
and  closer  to  the  undulating  border.  Posterior  to  it  is  the  smaller 
and  more  deeply  stained  blepharoplast.  Close  to  the  latter  a 
small  clear  colorless  area  is  commonly  seen.  The  flagellum 


SPECIFIC   MICRO-ORGANISMS 


* 


FIG.  148. — Trypanosoma  rotatorium  in  blood  of  a  frog;  drawn  from  a  preparation 
stained  by  Romawowsky  method  after  dry  fixation.  The  smaller  form  is  feebly 
stained. 


FIG.  149. — Trypanosoma  rotatorium.  The  various  forms  which  occur  in  arti- 
ficial culture.  A,  Crithidia  form;  B,  trypanosome  form;  C,  spherical  form;  D  and 
E,  club  forms;  F  and  G,  spirochete  forms;  H,  resting  stage;  /,  resting  stage  with 
vacuole  and  double  nucleus.  (After  Doflein.) 


MASTIGOPHORA 


originates  near  the  blepharoplast  and  extends  along  the  convex 
border  of  the  cell,  which  is  drawn  out  into  a  well-developed  thin 
undulating  membrane,  to  the  anterior  end  of  the  cell  and  beyond 
it  as  a  free  flagellum.  The  posterior  tip  of  the  cell  is  usually 
drawn  out  to  form  a  slender  process.  The  other  border  of  the 
cell  is  nearly  straight  and  the  cytoplasm  near  it  usually  shows 
definite  evidence  of  longitudinal 
striation,  indicating  the  presence 
of  elementary  muscular  structures, 
so-called  myonemes.  The  slender 
form  resembles  very  closely  the 
shape  of  mammalian  trypano- 
somes. 

Cultures  of  Tr.  rotatorium  were 
first  obtained  by  Lewis  and  H.  U. 
Williams  in  the  condensation  fluid 
of  slanted  blood-agar.  Various 
forms  of  the  organism  occur  in 
the  cultures.  Many  of  these  are 
doubtless  degenerating  cells.  The 
mode  of  transmission  from  frog  to 
frog  is  unknown  but  it  is  prob- 
ably accomplished  by  means  of  leeches. 

Trypanosoma  Lewisi. — This  organism,  the  common  rat 
trypanosome,  appears  to  have  been  seen  as  early  as  1845,  but  its 
modern  study  dates  from  its  rediscovery  by  Lewis  in  1879.  It 
occurs  in  the  blood  of  wild  rats  throughout  the  world,  from  i  to 
40  per  cent  being  infected.  In  the  rat  the  parasite  passes  through 
a  short  period,  8  to  14  days,  of  rapid  multiplication,  which  is 
followed  by  a  period,  usually  several  weeks  or  months,  in  which 
the  organism  persists  without  evident  increase  in  numbers; 
further  multiplication  beginning  upon  transfer  to  a  new  host.  In 
the  adult  or  resting  stage,  the  trypanosomes  are  quite  uniform, 
1.5  to  2fjL  wide  by  27  to  28/z  in  length,  including  the  flagellum 
(Fig.  150).  When  blood  containing  these  adult  forms  is  injected 


FIG.    150. — Trypanosoma   lewisi.     X 
2500.     (From  Doflein  after  Minchin.) 


382, 


SPECIFIC  MICRO-ORGANISMS 


into  a  healthy  young  rat  the  multiplication  forms  of  the  parasite 
appear  after  about  three  days.  These  forms  show  a  great  variety 
of  size  and  shape  and  they  stain  more  deeply  than  the  adult  stage 
(Fig.  151).  Numerous  dividing  parasites  are  also  present,  some  of 
them  showing  multiple  division  with  the  formation  of  rosettes. 
The  division  is  longitudinal  and  essentially  unequal,  as  one  cell 
retains  the  old  flagellum  while  the  new  one  is  formed  for  the  other 


I 


FIG.  151. — Trypanosoma  lewisi.    Various  forms  in  the  blood  of  a  rat  six  days  after 
inoculation.     X  1125.     (After  MacNeal.) 

daughter  cell.  The  rosettes  arise  by  successive  longitudinal 
divisions,  and  an  unbroken  rosette  contains  one  cell  with  the  old 
flagellum  larger  than  the  others  (Fig.  152). 

The  infection  is  readily  transmitted  to  young  rats  by  the 
injection  of  blood  containing  the  parasites.  Under  natural  condi- 
tions transmission  is  due  to  insects,  especially  fleas  and  lice.1  The 

1  Swellengrebel  and  Strickland:  Parasitology,  1910,  Vol.  Ill,  pp.  360-389. 


MASTIGOPHORA 


383 


trypanosomes  multiply  in  the  digestive  tract  of  these  insects, 
producing  various  forms,  many  of  them  resembling  herpetomonas 
and  leptomonas.  Fleas  remain  infective  for  a  long  time. 

Cultures  of  Tr.  lewisi  were  obtained  by  MacNeal  and  Novy1 
in  1902-03,  in  the  condensation  fluid  of  inclined  blood- agar,  and 
the  infection  was  reproduced  by  inoculation  of  these  cultures. 


FIG.  152. — Trypanosoma  lewisi.  Eight-cell  rosette  in  division.  Note  the  long 
original  or  parent  whip  on  one  of  the  cells.  Several  cells  show  a  second  flagellum 
growing  out  preparatory  to  a  further  division.  X225o.  (After  MacNeal.) 

The  size  and  shape  of  the  organism  in  culture  is  quite  variable 
The  actively  dividing  forms  are  usually  grouped  in  rosettes  with 
flagella  directed  centrally,  and  the  cells  themselves  are  pear- 
shaped  or  oval.  Herpotomonad  forms  are  common. 

The  infection  with  Tr.  lewisi  rarely  results  in  death  of  the  rat. 

1  Contributions  to  Medical  Research,  dedicated  to  Victor  Clarence  Vaughan, 
1903,  PP.  549-577- 


384 


SPECIFIC   MICRO-ORGANISMS 


Other  species  of  animals  are  not  readily  infected.  Immunity 
follows  recovery.  Artificial  immunity  has  been  produced  by 
Novy,  Perkins  and  Chambers1  by  the  injection  of  a  pure  culture 
which  had  been  propagated  for  six  years  on  artificial  media  and 
had  lost  its  virulence. 

There  are  many  other  relatively  harmless  trypanosomes 
parasitic  in  the  blood  of  various  mammals. 

Trypanosoma  Brucei. — Bruce  in  1895  discovered  this  organism 


D 


FIG.  153.  —  The  most  important  trypanosomes  parasitic  in  vertebrates.  A, 
Tr.kwisi;  B,  Tr.  eiansi  (India);  C,  Tr.  evansi  (Mauritius);  D,  Tr.  brucei;  E,  Tr. 
equiperdum;  F,  Tr.  equinium;  G,  Tr.  dimorphon;  H,  Tr.  gambiense.  All  magnified 
(From  Do  flein  after  Novy.} 


in  the  blood  of  horses  suffering  from  Nagana,  the  Tsetse-fly  dis- 
ease of  Zululand.  Pure  cultures  have  been  obtained  in  the  con- 
densation fluid  of  inclined  blood-agar  by  Novy  and  MacNeal 
and  the  injection  of  pure  cultures  into  animals  produces  the  dis- 
ease and  death. 

Tr.  brucei  is  1.5  to  5ju  wide  and  25  to  35/z  long,  including  the 

1  Journ.  Inf.  Dis.y  1912,  Vol.  XI,  pp.  411-426. 


MASTIGOPHORA 


385 


flagellum.  The  nucleus  lies  near  the  center  of  the  cell.  It  is  oval 
or  somewhat  irregular  in  outline  and  usually  occupies  the  who4e 
width  of  the  cell.  Near  the  blunt  posterior  end  of  the  cell  is  a 

A 


FIG.  154. — Glossinamorsitaus.     A,  Magnified.    (After  Dofiein.}     B,  Sketch  showing 
natural  size.     (From  Dofiein  after  Blanchard.} 

spherical  granule,   the  blepharoplast.     Near   this  the  flagellum 
originates  and  it  extends  forward  along  the  convex  border  of  the 
cell,  which  is  drawn  out  into  a  thin  undulating  membrane,  and 
A  B 


V 

FIG.  155. — Glossina  morsitaus-,  lateral  view  of  the  resting  fly.     A,  Before  feeding. 
B,  After  sucking  blood.     (From  Doflein  after  Austen.} 

extends  beyond  the  anterior  end  of  the  cell  as  a  free  flagellum. 
The  cytoplasm  anterior  to  the  nucleus  often  contains  many  coarse 
granules.  The  general  shape  of  the  trypanosome  as  seen  in  the 

25 


386  SPECIFIC   MICRO-ORGANISMS 

blood  of  the  infected  animal  is  fairly  uniform.  There  is,  however, 
considerable  variety  in  size,  internal  structure  and  staining 
properties.  Multiplication  takes  place  by  unequal  longitudinal 
division,  much  the  same  as  in  Tr.  lewisi,  but  the  dividing  cell  has 
the  same  general  form  as  the  others  and  multiple  division  figures 
are  less  common.  The  larger  cells  are  usually  in  process  of  divi- 
sion. Trypanosomes  with  feebly  staining  cytoplasm  and  others 
with  very  abundant  coarse  granules  also  occur.  The  former  are 
probably  degenerating  and  disintegrating  cells. 

Tr.  brucei  is  taken  up  by  the  blood-sucking  tsetse  fly,  Glossina 
morsitans  and  in  about  5  per  cent  of  these  it  multiplies  in  the 
alimentary  canal  and  penetrates  into  the  body  cavity,  causing  a 
generalized  infection  of  the  fly.  After  about  three  or  four  weeks 
the  salivary  glands  are  invaded  and  the  fly  is  then  able  to  infect 
other  animals  by  biting  them,  and  it  remains  infective  for  a  long 
time,  probably  as  long  as  it  lives.  Other  insects  may  possibly 
serve  to  transmit  the  parasite.  The  infection  is  also  readily 
transmitted  from  animal  to  animal  by  the  injection  of  infected 
blood. 

Cultures  are  obtained  with  some  difficulty,  but  most  readily 
by  inoculating  inclined  blood-agar,1  2:1,  and  incubating  at  28°  C. 
The  primary  cultures  should  not  be  transplanted  until  they  are 
about  three  weeks  old,  and  they  usually  fail  to  infect  animals  if 
injected  into  them.  The  virulence  is  regained  in  the  subcultures. 
Culture  filtrates  are  not  toxic.  The  poison  of  trypanosomes 
seems  to  be  set  free  as  a  result  of  their  disintegration  in  the  body 
fluids.2 

Nagana  occurs  naturally  in  a  great  variety  of  the  quadrupeds 
and  is  usually  fatal.  Man  is  not  susceptible.  Mice  and  rats 
die  in  6  to  14  days  after  inoculation.  Guinea-pigs  may  show  one 
or  more  relapses,  the  disease  lasting  for  two  to  ten  weeks. 

1  The  agar  employed  should  contain  the  extractives  of  125  grams  of  meat,  10  grams 
pepton,  5  grams  salt  and  25  grams  of  agar  in  1000  c.c.  It  is  liquefied,  cooled  to 
50°  C.  and  mixed  with  twice  its  volume  of  warm  defibrinated  rabbit's  blood  and  then 
allowed  to  solidify  in  an  inclined  position. 

2MacNeal:  Journ.  Inf.  Dis.,  1904,  Vol.  I,  p.  537. 


MASTIGOPHORA 


387 


Diagnosis  may  be  made  by  microscopic  examination  of  the 
blood  when  the  parasites  are  numerous.  At  other  times  it  is  well 
to  inject  5  to  10  c.c.  of  blood  into  a  white  rat.  The  distinction 
of  Tr.  brucei  from  other  species  of  trypanosomes  causing  similar 
diseases  is  not  easy  and  may  require  prolonged  study. 

Immunity  of  susceptible  animals  has  not  yet  been  achieved, 
but  inoculation  with  attenuated  cultures  produces  a  relative 
immunity  in  small  laboratory  animals.1 

Trypanosoma  Evansi. — This  organism  was  discovered  by 
Griffith  Evans  in  1880  in  the  blood  of  horses  and  various  other 


FIG.  156. — Trypanosoma  equiperdum.     Blood  of  an  inoculated  rat.     A,  after  four 
days;  B,  after  eight  days.     (After  Doflein.} 

animals  suffering  from  the  disease  known  in  India  as  Surra.  The 
trypanosome  resembles  Tr.  brucei  in  most  respects  but  is  recog- 
nized as  a  distinct  species.  Surra  is  apparently  transmitted  by 
various  flies,  Tabanidcz,  Stomoxys,  and  also  by  fleas. 

Trypanosoma  equiperdum  was  found  by  Rouget  in  1896  in 
the  blood  of  horses  suffering  from  dourine.  The  infection  is 
transmitted  by  coitus  and  probably  also  in  other  ways.  Dourine 
occurs  in  southern  Europe  and  northern  Africa.  A  few  cases 
have  been  observed  in  Canada  and  in  the  United  States.  Small 
laboratory  animals  are  susceptible  to  inoculation. 

1  Novy,  Perkins  and  Chambers:  Journ.  Inf.  Dis.,  1912,  Vol.  XI,  pp.  411-426. 


388  SPECIFIC   MICRO-ORGANISMS 

Trypanosoma  Equinum. — Elmassian  in  1901  observed  this 
organism  in  the  blood  of  horses  suffering  from  Mai  de  Caderas 
in  South  America.  It  possesses  a  very  minute  blepharoplast,  a 
morphological  character  which  distinguishes  it  from  most  other 
trypanosomes.  Small  laboratory  animals  are  susceptible. 

Several  other  species  of  trypanosomes  have  been  described, 
which  cause  fatal  diseases  in  quadrupeds.  Most  of  these  have 
been  found  in  Africa. 

Trypanosoma  Gambiense. — Button  and  Todd  in  1901  ob- 
served this  organism  in  the  blood  of  an  Englishman  in  Gambia. 
The  parasite  had  been  previously  seen  by  Forde.  The  disease, 
which  resulted  in  death  after  two  years,  was  called  trypansoma 
fever.  Castellani  in  1903  observed  trypanosomes  in  the  cerebro- 
spinal  fluid  of  patients  suffering  from  sleeping  sickness  in  Uganda. 
This  organism  is  now  known  to  be  the  same  as  the  Tr .  Gambiense 
of  Button,  and  sleeping  sickness  is  recognized  as  the  terminal 
stage  of  trypanosoma  fever. 

Tr.  gambiense  is  very  similar  in  form  to  Tr.  brucei  but  the 
posterior  end  is  on  the  average  somewhat  more  pointed.  The 
length  varies  between  15  to  30^  and  the  width  from  i  to  3/z.  The 
significance  of  the  different  forms  found  in  the  blood  is  not  defi- 
nitely known.  Multiplication  takes  place  in  the  same  way  as 
in  Tr.  brucei.  In  the  tsetse  fly,  Glossina  palpalis,  the  trypano- 
somes slowly  disintegrate  and  disappear  during  the  first  four  days 
after  the  infected  blood  is  ingested,  and  in  most  of  the  flies  this 
results  in  extermination  of  the  trypanosomes.  In  5  to  10  per 
cent  of  the  flies  the  parasites  are  not  completely  destroyed,  but 
the  early  diminution  in  their  number  is  followed  by  an  abundant 
multiplication  of  the  trypanosomes  in  the  stomach  and  intestine  of 
the  insect.  After  18  to  53  days  these  flies  become  capable  of 
infecting  new  animals  by  their  bite  and  remain  infectious  for  a 
very  long  time.  The  parasites  are  found  in  the  salivary  glands1 
when  the  fly  becomes  capable  of  causing  the  disease.  A  great 

1  Bruce,  Hamerton,  Bateman  and  Mackie:  Proc.  Royal  Soc.,  1911,  Ser.  B, 
Vol.  LXXXIII,  pp.  338-344;  PP.  345-348;  pp.  513-527- 


MASTIGOPHORA  389 

diversity  of  form  is  observed  in  the  trypanosomes  within  the  fly  but 
the  significance  of  the  different  types  is  not  yet  fully  understood. 

Many  of  the  mammals  are  susceptible  to  inoculation  with 
Tr.  gambiense.  White  rats  usually  relapse  2  or  3  times  before 
finally  succumbing  to  the  infection,  whereas  they  usually  die 
within  2  weeks  when  inoculated  with  Tr.  brucei.  The  virulence 
of  the  organism  is  somewhat  variable. 

Attempts  to  cultivate  Tr.  gambiense  in  artificial  media  have 
not  been  fully  successful.  It  has  been  possible  to  obtain  multipli- 


FIG.  157. — Glossina  palpalis  in  natural  resting  position,  and  with  wings  outstretched. 

(After  Dofiein.} 

cation  of  the  organisms  and  to  keep  them  alive  for  several  weeks 
on  blood-agar  but  such  cultures'are  not  virulent  and  cannot  be 
kept  up  indefinitely.1 

Human  trypanosomiasis  is  a  most  important  and  widespread 
disease  in  equatorial  Africa.  Symptoms  appear  long  after  the 
infection  has  taken  place.  The  disease  manifests  itself  in  two 
forms,  the  trypanosoma  fever  and  the  sleeping  sickness.  Trypano- 
soma  fever  is  an  irregularly  remittent  fever  lasting  for  several  days 
at  each  attack,  accompanied  by  a  macular  eruption,  and  always 

1  Thomson  and  Sinton:  Annals   of  Trop.  Med.   and  ParasitoL,  1912,  Vol.  VI, 
PP-  331-356. 


390  SPECIFIC   MICRO-ORGANISMS 

associated  with  a  general  enlargement  of  the  lymph  nodes.  The 
trypanosomes  are  numerous  in  the  blood  during  the  febrile  period 
and  become  very  scarce  during  the  intermissions.  The  fever 
leads  to  emaciation  and  death,  sometimes  without  inducing  the 
terminal  coma  and  sometimes  with  the  production  of  typical 
sleeping  sickness.  The  sleeping  sickness  is  characterized  by 
prolonged  coma  and  progressive  emaciation.  At  intervals  the 
patient  may  be  aroused  and  given  nourishment,  but  eventually 
this  is  no  longer  possible.  At  this  stage  the  trypanosomes  are 
present  in  the  cerebrospinal  fluid.  Bacterial  infection  of  the 
meninges  often  takes  place  as  a  terminal  event.  It  is  conserva- 


FIG.  158. — Trypanosoma  amum  in  the  blood  of  common  wild  birds.     X    1500. 
(After  Novy  and  MacNeal.) 

tively  estimated  that  100,000  natives  have  died  of  trypanosomiasis 
in  Africa  from  1900  to  1910.  There  have  been  several  cases  in 
Europeans.  Recovery  seems  to  be  rather  uncommon  but  does 
occur. 

Trypanosoma  Rhodesiense. — Stephens  and  Fantham1  have 
studied  a  case  of  human  trypanosomiasis  contracted  in  north- 
eastern Rhodesia,  where  Glossina  palpalis  does  not  occur.  The 
parasite  differs  somewhat  from  Tr.  gambiense  and  is  regarded  by 

1  Proc.lRoyal  Soc.,  1910,  Ser.  B,  Vol.  LXXXIII,  pp.  28-33. 


MASTIGOPHORA 


391 


these  authors  as  a  distinct  species.     It  seems  to  be  transmitted 
by  Glossina  morsitans.1 

Trypanosoma  Avium. — Trypanosomes  were  probably  seen  in 
the  blood  of  birds  by  earlier  investigators,  but  the  first  accurate 
description  of  such  observations  is  that  of  Danilewsky  in  1885. 


FIG.  159. — Trypanosoma  avium  in  culture  on  blood  agar.     X  1500.     (After  Novy 

and  MacNeal.} 

Infection  with  trypanosomes  is  very  common  in  the  ordinary 
wild  birds.  Novy  and  Mac  Neal2  examined  431  American  birds 
representing  40  common  species  and  found  trypanosomes  in  38 
individuals,  representing  16  species.  The  indicated  prevalence 

1  Kinghorn  and  Yorke:  Annals  of  Trop.  Med.  and  ParasitoL,  1912,  Vol.  VI, 
pp.  269-285.     Kinghorn,  Yorke  and  Lloyd:  ibid.,  1912,  Vol.  VI,  pp.  495-503. 
2Journ.  Infect.  Dis.,  1905,  Vol.  II,  pp.  256-308. 


3Q2  SPECIFIC   MICRO-ORGANISMS 

of  the  infection,  8.8  per  cent,  is  doubtless  far  below  the  actual 
percentage,  as  many  of  the  birds  were  not  tested  by  the  cultural 
method.  There  are  doubtless  several  species  of  bird  trypanosomes 
but  the  most  common  form  is  Tr.  avium.  The  length  varies 
from  25  to  yo/x  and  the  width  from  4  to  yju. 

Cultures  are  easily  obtained  by  transferring  the  infected  blood 
to  tubes  of  blood-agar  and  incubating  at  25°  to  30°  C.  The  pro- 
tozoa grow  abundantly  and,  by  weekly  transfers,  may  be  kept 
under  cultivation  without  special  difficulty  for  an  indefinite  period. 
Injection  of  cultures  into  birds  is  only  rarely  followed  by  appear- 
ance of  trypanosomes  in  the  blood. 

The  parasites  persist  in  the  blood  of  the  birds  for  many  months 
and  probably  for  years.  They  seem  to  be  comparatively  harmless. 
The  mode  of  transmission  from  bird  to  bird  in  unknown. 

Trypanosoma  avium  is  a  form  of  considerable  importance  in 
the  study  of  systematic  protozoology  because  of  the  confusion  of 
trypanosomes  and  hemocytozoa  by  Schaudinn1  in  1904,  who 
regarded  Tr.  avium  as  merely  an  extracellular  form  of  Hamopro- 
teus  noctucB  (danilewskyi?)  (see  page  414).  This  misconception, 
together  with  the  analogous  assumption  of  similar  relationship 
between  spirochetes  of  birds  and  the  leukocytozoon  of  Ziemann, 
Hczmoproteus  ziemanni,  made  by  Schaudinn  at  the  same  time, 
has  exercised  a  profound  influence  upon  the  course  of  investiga- 
tion in  the  groups  of  spirochetes,  trypanosomes  and  hemocytozoa 
during  the  last  eight  years,  and  it  is  only  recently  that  this  error 
of  Schaudinn  has  been  recognized  as  such  by  the  German  and 
English  protozoologists. 

Schizotrypanum  Cruzi. — Chagas  discovered  this  organism 
in  1907.  It  occurs  in  the  blood  in  the  Brazilian  human  trypano- 
somiasis  called  coreotrypanosis.  Multiplication  takes  place 
within  endothelial  cells,  lymphocytes  and  other  cells  in  the  paren- 
chymatous  organs,  and  especially  in  the  interior  of  muscle  cells  in 
the  heart  and  skeletal  muscles. 2  The  dividing  parasites  are  without 

1  Arb.  a.  d.  Kais.  Gesundheitsamte,  1904,  Vol.  XX,  pp.  387-439. 
2Vianna:  Memorias  do  Institute  Oswaldo  Cruz,   1911,  Vol.  Ill,  pp.   276-293. 
Abstract  in  Sleeping  Sickness  Bull.,  1912,  Vol.  IV,  pp.  288-293. 


MASTIGOPHORA 


393 


FIG.  1 60. — Schizotrypanum  cruz'i  developing  in  the  tissues  of  the  guinea-pig, 
i.  Cross-section  of  a  striated  muscle  fiber  containing  Schizotrypanum  cruzi:  Note 
dividing  forms.  2.  Section  of  brain  showing  a  Schizotrypanum  cyst  within  a 
neuroglia  cell,  containing  chiefly  flagellated  forms.  3.  Section  through  the  supra- 
renal capsule,  fascicular  zone.  4.  Section  of  brain  showing  a  neuroglia  cell  filled 
with  round  forms  of  Schizotrypanum.  (From  Low,  in  Sleeping  Sickness  Bulletin, 
after  Vianna.} 


394  SPECIFIC   MICRO-ORGANISMS 

flagella  and  resemble  the  intracellular  forms  of  Leishmania.  From 
these  cysts  the  parasites  escape  into  the  blood,  where  they  are 
found  as  trypanosomes  in  the  blood  plasma.  Slender  and  thick 
forms  occur  here,  the  difference  probably  depending  upon  the 
age  of  the  parasites. 

Monkeys,  rats,  mice,  young  guinea-pigs  and  many  other 
mammals  are  susceptible  to  inoculation.  The  infection  is  trans- 
mitted by  a  bug,  Conorhinus  megistus, 
in  which  the  protozoon  develops 
abundantly.  The  bedbug,  Culex 

lectularius   also   is  capable  of  trans-   kj?  C\     £*~" 

mitting  the  disease.  f*r\  *\^' 

Cultures    are    readily    obtained  %4^      ' 
on    blood-agar     and     Chagas    was        pIG>     ,  6 1  .-Schizotrypanum 
able  to  infect  animals  with  such  cul-   cruzi   in    human   blood.     (From 

Doflein  after  Chagas.) 

tures. 

Leishmania  Donovani. — Laveran  and  Mesnil  in  1903  described 
this  protozoon  which  occurs  inside  cells  in  various  parts  of  the 
body,  but  is  especially  abundant  in  the  spleen  and  liver,  in  the  dis- 
ease known  in  India  as  Kala-Azar  or  tropical  splenomegaly.  The 
organism  is  oval,  2  to  4/4  in  diameter,  finely  granular  and  some- 
times vacuolated.  In  the  interior  there  is  a  large  rounded  nucleus 
and  a  smaller  oval  or  rod-shaped  blepharoplast,  near  which  a  third 
very  slender  short  thread  may  usually  be  recognized  as  the  rudi- 
ment of  the  undeveloped  flagellum.  These  structures  are  doubled 
in  the  division  stages.  Multiple  division  also  occurs.  In  the  cir- 
culating blood  the  organism  is  found  within  lymphocytes  and  poly- 
nuclear  leukocytes.  Many  of  them  may  be  found  in  a  single  cell. 

Cultures  are  readily  obtained  by  inoculating  fluid  (citrated) 
blood  with  blood  or  with  spleen  juice  containing  the  parasites,  or 
by  inoculating  the  usual  blood-agar.  In  artificial  culture  the 
cell  elongates,  the  rudimentary  whip  extends  into  a  true  flagellum 
and  the  organism  assumes  the  appearance  of  a  typical  leptomonas 
(herpetomonas).  Little  difficulty  is  experienced  in  keeping  the 
cultures  alive  and  flourishing. 


MASTIGOPHORA 


395 


The  parasite  has  been  supposed  to  be  transmitted  from  man 
to  man  by  bugs  of  the  genus  Cimex,  but  this  hypothesis  has  be_en 


FIG.  162. — Conorhinus  megistus,  the  insect  carrier  of  Schizotrypanum  cruzi.    (From 

Dofltin  after  C  ha  gas.} 


*•* 


FIG.  163. — Lieshmania  donovani  in  the  juice  obtained  by  puncture  of  the  spleen  in 
kala-azar.     (From  Doflein  after  Donovan.} 

rendered  very  uncertain  by  recent  work  of  Wenyon1  and  the 

1  Journ.  Lond.  Sch.  Trop.  Med.,  1912,  Vol.  II,  pp.  13-26. 


396 


SPECIFIC   MICRO-ORGANISMS 


Sergents.1     The  latter  investigators  were  able  to  effect  experi- 
mental transmission  by  means  of  the  dog  flea,  Ctenocephalus  canis. 

Kala-Azar  is  'endemic  in  tropical  Asia 
and  northeast  Africa,  where  it  occurs  among 
the  poorer  class  of  people,  living  in  squalor. 
It  is  characterized  by  irregular  fever,  weak- 
ness and  cachexia  and  especially  by  enormous 
enlargement  of  the  spleen,  often  of  the  liver 
also.  It  is  frequently  fatal.  Dogs  and  mon- 
keys are  susceptible  to  inoculation. 

LeishmaniaTropica. — This  organism  was 
first  accurately  described  by  J.  H.  Wright,2 
who   found  it  in   great   abundance  in   the 
lesion   known   as   Aleppo    boil,    Delhi    boil    or    tropical    ulcer. 
The  parasites  occur  within  the  endothelial  cells  within  the  lesion 


FIG.  164. — Leishmania 
donovani,  various  forms 
observed  in  artificial  cul- 
ture. (From  Doflein  after 
Chatter jee.) 


IM 


FIG.  165. — Leishmania  tropica.     Smear  from  a  Delhi  boil.     Xisoo.     (From  Doflein 

after  J.  H.  Wright.} 

and  are  very  numerous.     Leishmania  tropica  resembles  L.  donovani 

1  Sergent  (Edm.  &  Et.),  L'Heritier  and  Lemaire,  Bull.  Soc.  Path.  Exot.,  1912, 
Vol.  V,  pp.  595-597- 

2  Journ.  Med.  Rsch.,  1903,  Vol.  X,  pp.  472-482. 


MASTIGOPHORA 


397 


very  closely  except  in  its  pathogenic  properties.  Cultures  on 
blood-agar  have  been  obtained  by  Nicolle  and  are  easily  propa^ 
gated  at  22°  C.  Dogs  and  monkeys  are  susceptible  to  inoculation 
and  the  human  disease  is  probably  contracted  from  dogs  through 
the  agency  of  insects.  The  disease  is  relatively  benign  and  recov- 
ery is  followed  by  prolonged  immunity.  Inoculation  has  been 
practised  in  man  in  order  to  produce  immunity. 


FIG.  166. — Lciskmania  Iropica, 
forms  observed  in  cultures.  (From 
Dofiein  after  Nicolle.) 


FIG.  167. — Try pano  plasma  cy- 
prini.  Bl,  Blepharoplast;  N,  nu- 
cleus. X  2000.  (After  Doflein.} 


Leishmania  Infantum. — Nicolle  in  1908  observed  this  organ- 
ism in  the  spleen,  liver  and  bone  marrow  of  children  dying  from 
splenomegaly  in  northern  Africa.  The  disease  resembles  Kala- 
Azar  in  all  respects  except  that  the  patients  are  all  very  young. 
Dogs  are  naturally  infected  with  this  parasite  and  are  probably 
the  source  of  the  human  disease.  Cultures  on  blood-agar  are 
readily  obtained  and  kept  up  indefinitely  without  special  difficulty. 


398 


SPECIFIC  MICRO-ORGANISMS 


Trypanoplasma  Borreli. — Laveran  and  Mesnil  in  1901  de- 
scribed this  protozoon  which  occurs  in  the  blood  of  various  species 
of  fish.  It  resembles  a  trypanosome  somewhat,  but  the  blepharo- 
plast  is  relatively  large  and  from  it  two  flagella  originate,  one 
extending  forward  immediately  as  a  free  whip  while  the  other 
runs  along  the  convex  border,  ensheathed  in  an  undulating  mem- 
brane, and  extends  at  the  posterior  end 
as  a  free  flagellum.  Longitudinal  divi- 
sion takes  place  in  the  circulating  blood. 
Transmission  seems  to  be  accomplished 
by  means  of  leeches.  T.  cyprini  and 
T.  guernei  seem  to  be  identical  with  T. 
borreli,  but  they  may  prove  to  be  dis- 
tinct species. 


FIG.  1 68. — Bodo  lacertce.  a, 
Sketched  from  life;  b,  drawn  from 
a  stained  preparation.  (From 
Doflein  after  H  artmann  and 
Prowazek.} 


FIG.  169. — Trichomonas  hominis  from  the 
mouth.     (From  Doflein  after  Prowazek.) 


Bodo  Lacertae. — In  the  cloaca  of  various  lizards  a  flagellate 
is  almost  constantly  found.  It  is  2  to  4^  wide  and  6  to  12.5^ 
long,  lance-shaped  and  twisted  at  the  posterior  (pointed)  end. 
The  nucleus  is  near  the  anterior  end.  At  its  side  is  a  granule 
resembling  a  blepharoplast  and  from  this  a  thread  extends  to 
the  anterior  end  of  the  cell  where  it  gives  rise  to  two  flagella. 


MASTIGOPHORA 


399 


FIG.  170. — Lamblia  intestinalis.  A,  Ventral  aspect;  B,  lateral  view;  C,  in  posi- 
tion on  epithelium;  D,  the  same  enlarged.  (From  Doflein  after  Grassi  and  'Sckevria- 
kof.) 

A  B  C 


s 


FIG.  171. — Trimastigamceba  philippinensis.  A,  Early  stage  of  division  of  the 
nucleus.  The  polar  caps  are  still  united  by  a  bridge.  The  equatorial  plate  has 
formed.  B,  Ordinary  cyst.  C,  Vegetative  form  showing  the  nucleus  and  a  second 
chromatin  granule  (split  off  from  it?).  D,  Flagellated  form  showing  remains  of  the 
rhizoplast  between  the  nucleus  and  the  basal  granules.  £,  Flagellated  form  with 
pseudopodia.  (After  Whitmore.} 


4OO  SPECIFIC   MICRO-ORGANISMS 

Trichomonas  Hominis. — Davaine  observed  this  parasite  in 
1854.  It  is  common  in  the  human  digestive  tract,  especially  in 
the  stomach  in  anacidity  and  in  the  intestine  in  chronic  digestive 
disturbances.  The  organism  is  3  to  4/z  wide  and  4  to  i5/z  long, 
pear-shaped  and  provided  with  three  free  flagella,  and  a  fourth 
thread  which  passes  around  one  side  of  the  cell  in  the  margin  of 
the  undulating  membrane.  The  parasite  seems  to  be  a  harmless 
commensal,  as  a  rule,  but  it  may  possibly  bear  some  causal  rela- 
tion to  diarrhea  in  some  cases.  Animals  have  not  been  success- 
fully inoculated  with  it.  Tr .  vaginalis  is  very  similar.  It  grows 
in  the  acid  vaginal  mucus.  Other  trichomonad  forms  occur  in 
the  intestines  of  animals,  particularly  in  mice,  in  frogs  and  in 
lizards. 

Lamblia  Intestinalis.- — The  cell  has  the  form  of  a  turnip  with 
a  wide  and  deep  excavation  in  front  near  the  anterior  rounded 
end,  forming  a  suction  cup.  The  body  is  bilaterally  symmetrical. 
The  length  is  10  to  2i/z  and  the  width  5  to  i2ju.  There  are  eight 
flagella,  each  from  9  to  14/1  long.  The  mode  of  multiplication  is 
not  fully  known.  Resistant  cysts  are  formed,  probably  after 
sexual  union  of  two  individuals,  and  these  escape  with  the  feces 
and  lead  to  the  infection  of  new  hosts.  Lamblia  lives  in  the  duo- 
denum and  jejunum  of  man  and  many  other  mammals.  It  ap- 
pears to  be  relatively  harmless  in  most  cases  but  the  possibility 
that  it  may  be  a  cause  of  digestive  disturbance  must  be  con- 
sidered. It  is  often  present  in  chronic  dysenteries. 

Mastigamceba  Aspera. — This  a  saprophytic  form,  described 
by  Schulze,  which  possesses  a  single  flagellum,  but  is  also  capable 
of  extending  finger-like  projections  of  its  cytoplasm,  pseudopodia, 
just  as  an  ameba  does.  Whitmore1  has  described  a  somewhat 
similar  saprophyte,  Trimastigamceba  philippinensis,  which  is  at 
times  ameboid  without  flagella  and  at  other  times  possesses  three 
or  possibly  four  whips.  It  divides  and  encysts  like  an  ameba. 
The  organism  is  readily  cultivated  on  the  alkaline  agar  of  Mus- 
grave  and  Klegg. 

1  Archivf.  Prolistenkunde,  1911,  Bd.  XXIII,  S.  81-95. 


CHAPTER  XXVIII. 
RHIZOPODA. 

Amoeba  Proteus. — This  large  saprophytic  ameba  may  be 
considered  as  an  example  of  the  numerous  species  of  free-living 
amebae,  the  classification  and  identification  of  which  is  still  in 
hopeless  confusion.  The  organism  is  widely  distributed  in  stag- 
nant water  and  is  easily  cultivated  in  the  laboratory  in  not  too 
foul  infusions  containing  bacteria  and  algae.  The  cell  is  50  to 


FIG.  172.  —  A,  Amoeba  proteus  engulfing  a  clump  of  small  alg<2  (No).  Cv,  con- 
tractile vacuole;  N,  nucleus.  B,  Newly  encysted  ameba  showing  nuclear  fragments; 
cy,  cyst  wall;  w,  nucleus;  R,  reserve  food  substance.  C,  Cyst  containing  man}''  young 
amebaj  beginning  to  escape;  cy,  cyst  wall;  k,  young  amebae.  (After  Doflein.) 


across,  often  possesses  numerous  thick,  blunt  pseudopodia. 
The  ectoplasm  and  endoplasm  appear  distinctly  different,  the 
latter  being  filled  with  granules,  crystals,  vacuoles  and  food  parti- 
cles, such  as  algae  and  bacterial  cells,  and  possessing  a  contractile 
vacuole.  The  nucleus  is  lentil-shaped  and  the  chromatin  within 
it  has  a  very  typical  arrangement  in  a  central  plate  surrounded 
by  a  network  on  which  the  peripheral  chromatin  is  symmetrically 
26  401 


402  SPECIFIC   MICRO-ORGANISMS 

placed.  Binary  division  with  mitosis  of  the  nucleus  seems  to  be 
the  common  mode  of  multiplication.  Multiple  division  also 
occurs  in  the  vegetative  state.  The  resistant  stage  (cyst)  is  charac- 
terized by  a  thick,  firm  wall  of  several  layers,  within  which  the 
nucleus  divides  into  200  or  more  daughter  nuclei.  Each  of  these 
becomes  surrounded  by  a  little  cytoplasm  and,  when  the  cyst 
bursts,  wanders  out  as  a  young  ameba.  The  life  history  is  in- 
completely known. 

Cultures  of   saprophytic    amebae  are  readily  obtained  upon 
agar  plates.     The   medium  contains  agar  0.5  gram,  tap  water 


FIG.  173. — Eutamceba  coli.     a,  Free  ameba;  b,  ripe  cyst  with  eight  nuclei.     (From 

Doflein  after  Hartmann.) 

90  c.c.,  ordinary  nutrient  broth  10  c.c.  Cultures  are  incubated 
at  25°  C.  Williams1  has  succeeded  in  obtaining  pure  cultures, 
free  from  bacteria,  at  36°  C.  by  employing  agar  smeared  with 
naturally  sterile  brain  substance. 

Entamoeba  Coli. — Loesch2  in  1875  observed  amebae  in  the 
human  large  intestine  in  gastro-intestinal  disturbance.  The 
organism  is  very  common  in  the  human  intestine,  being  found  in 
10  to  60  per  cent  of  persons  without  digestive  disturbances, 
when  the  examination  is  thorough. 

The  cell  in  the  vegetative  stage  is  variable  in  shape  and  size, 

1  Journ.  Med.  Rsch.,  1911,  Vol.  XXV,  pp.  263-283. 

2  Virchow's  Archiv,  1875,  Bd.  LXV,  S.  196-211. 


RHIZOPODA  403 

the  diameter  measuring  10  to  70/1.  The  protoplasm  is  slightly 
granular  and  shows  distinctly  an  alveolar  structure.  The  d!s-~ 
tinction  between  ectoplasm  and  endoplasm  is  apparent  only  in 
the  pseudopodia.  There  is  no  contractile  vacuole.  Food  sub- 
stance is  present  in  the  cytoplasm,  bits  of  vegetable  material, 
bacteria  and,  rarely,  red  blood  cells.  The  nucleus  is  round,  ve- 
sicular and  enclosed  in  a  nuclear  membrane.  In  its  center  is  a 
relatively  large  mass  of  chromatin  and  there  are  numerous  smaller 
masses  of  chromatin  at  the  periphery  beneath  the  nuclear  mem- 
brane. Multiplication  in  the  vegetative  stage  takes  place  by 
binary  division  as  a  rule,  but  multiple  division  preceded  by  re- 
peated division  of  the  nucleus  also  occurs. 

E.  coli  discharges  all  food  material  from  its  cytoplasm  before 
encystment  so  that  the  cell  is  clear  and  the  nucleus  plainly  visible. 
A  large  vacuole  in  the  cytoplasm  usually  makes  its  appearance 
and  is  present  during  the  first  and  second  division  of  the  nucleus 
in  the  cyst.  It  is  large  in  those  cysts  in  which  much  chromatin 
escapes  from  the  nuclei  into  the  cytoplasm  as  chromidia,  and  it 
usually  disappears  when  the  four  nuclei  have  been  formed.  A 
further  division  of  the  nuclei  gives  rise  to  eight  and  this  is  the 
usual  number  present  in  the  fully  developed  cyst  of  E.  coli,  al- 
though rarely  ten  or  even  sixteen  nuclei  may  be  observed.1  The 
self-fertilization,  autogamy,  described  by  Schaudinn  as  occurring 
early  in  encystment  has  not  been  observed  by  Hartmann,  and 
its  actual  occurrence  seems  questionable.  The  developed  cyst 
with  eight  nuclei  is  about  i5/z  in  diameter  and  is  considered  to  be 
definitely  characteristic  of  this  species. 

E.  coli  is  generally  regarded  as  a  harmless  commensal  in  the 
human  intestine.  It  is  however  impossible  to  exclude  the  possi- 
bility that  it  may  contribute  to  the  aggravation  of  pathological 
conditions  present  in  the  digestive  tract.  (Compare  with  Bacillus 
coli.)  Its  common  occurrence  in  healthy  men  speaks  against 
its  possessing  any  very  specific  and  powerful  pathogenic  property. 

1  Hartmann   and  Whitmore:  Archiv  f.  Protistenkunde,    1912,   Bd.   XXIV,   S. 
182-194. 


404 


SPECIFIC   MICRO-ORGANISMS 


Entamceba  Tetragena. — Viereck  in  1906  recognized  this 
organism  as  a  species  distinct  from  E.  coli.  It  occurs  in  the  intes- 
tine and  in  the  stools  of  persons  suffering  from  amebic  dysentery 


FIG.  174. — Entamosba  telragena.     The  same  living  individual  drawn  at  brief  intervals 
while  moving.     (From  Doflein  after  Hartmann.) 

and  very  seldom  in  other  individuals.     The  cell  is  8  to  6o/i  in 
diameter.     The  ectoplasm  is  distinctly  differentiated  from  the 


FIG.  175. — Eutamceba  telragena.  a,  Vegetative  cell  containing  a  red  blood  cell 
(near  upper  end).  Xisoo.  b  and  c,  Drawings  of  nuclei  showing  stages  of  the  so- 
called  cyclical  changes.  X26oo.  (From  Doflein  after  Hartman.} 

endoplasm  even  when  the  cell  is  motionless,  and  the  lobose  pseudo- 
podia  are  made  up  entirely  of  the  stiff  highly  refractive  ectoplasm. 
The  endoplasm  contains  food  material  consisting  of  bacteria,  cell 


RHIZOPODA  405 

fragments  and  red  blood  cells.  The  nucleus  is  very  distinctly 
visible  in  the  living  ameba.  It  is  spherical  and  surrounded  by-a- 
thick  doubly  contoured  nuclear  membrane.  The  chromatin  is 
usually  distributed  just  beneath  the  nuclear  membrane  in  largest 
amount  and  in  the  center  there  is  a  karyosome  with  definite  centri- 
ole.  The  vegetative  multiplication  takes  place  by  division  into 
two  daughter  cells.  Multiple  division  seems  not  to  occur. 

Cyst  formation  is  rarely  observed.  The  cysts  are  most  likely 
to  be  found  when  the  stool  becomes  formed  in 
convalescence  from  an  attack  of  dysentery  and 
they  may  then  be  very  numerous.  The  mature 
cyst  contains  four  nuclei,  and  frequently  contains 
also  one  or  more  large  masses  of  chromidial  sub- 
stance which  stain  black  with  iron  hematoxylin. 


The  forms  of  the  organism  commonly  observed       FlG-  *  ib— 

.  £  .    ,  .  ,  ,  .        mceba  tetragena.  Ma- 

in the  feces  of  dysentery  are  either  the  active  ture  cyst  containing 
vegetative  cells1  or  degenerating  forms,  and  the  ^  STto^id 
latter  may  lead  to  confusion  unless  their  true  na-  substance.  (After 

j  Hartmann.) 

ture  is  recognized. 

E.  tetragena  is  regarded  as  the  causal  agent  of  amebic  or  tropical 
dysentery  and  there  can  be  little  question  that  it  is  the  parasite2 
present  in  most  cases  presenting  the  typical  clinical  picture  and 
pathology  of  the  disease.  It  is  doubtless  transmitted  in  food  and 
drinking  water  in  the  encysted  stage. 

Entamceba  Histolytica.  —  Schaudinn  in  1903  distinguished 
this  species  from  E.  coll  and  regarded  it  as  the  causal  organism 
in  amebic  dysentery.  The  subsequent  study  of  Schaudinn's 
preparations  by  Hartmann3  has  shown  that  most  of  the  specimens 
recognized  as  E.  histolytica  by  Schaudinn  are  in  reality  vegetative 
and  degenerating  forms  of  E.  tetragena.  Our  whole  knowledge 
of  the  species,  which  was  founded  upon  Schaudinn's  studies, 
therefore  becomes  very  uncertain  and  even  the  existence  of  E. 
histolytica  as  a  disticnt  species  may  be  seriously  questioned. 

1  Hartmann:  Arch.  f.  Protistenkunde,  1912,  Bd.  XXIV,  S.  163-181. 

2Whitmore:  Arch.  f.  Protistenkunde,  1911,  Bd.  XXIII,  S.  71-80. 

3  Hartmann,  in  Prowazek,  Handbuch  der  Path.  Protozoen,  1912,  Bd.  I,  S.  58-61. 


406  SPECIFIC  MICRO-ORGANISMS 

The  belief  that  amebae  bear  a  causal  relation  to  dysentery  is 
based  upon  the  fact  that  certain  types  of  amebae,  E.  tetragena 
(and  E.  histolytica?)  are  found  in  the  stools,  as  a  rule,  only  in 
cases  of  dysentery;  further,  that  these  cases  of  dysentery,  in 
which  these  amebae  occur,  are  characterized  by  definite  clinical 
signs  and  typical  anatomical  changes  in  the  intestine;  and  that 
these  amebae  are  found  penetrating  deeply  into  the  mucosa  of 
the  intestine,  and  it  is  possible  to  produce  ulcerative  enteritis 
in  experimental  animals  by  injecting  feces  containing  amebae 
into  the  rectum  or  by  feeding  fecal  material  containing  cysts;  and 
further,  the  fact  that  abscesses  occur  in  the  liver  in  amebic  dysen- 
tery, in  which  the  amebae  are  present  and  in  which  it  has  been 
impossible  to  demonstrate  the  presence  of  bacteria.  The  causal 
relation  seems  highly  probable,  but  it  must  be  recognized  that 
the  evidence  is  very  inconclusive  and  admits  of  other  possible 
explanations.  Even  the  relationships  of  the  various  forms  seen 
in  the  microscopic  preparations  require  a  certain  amount  of  specu- 
lation for  their  determination,  and  the  possibility  of  error,  even 
by  the  experienced  protozoologist,  must  be  recognized  and  has 
been  well  illustrated  by  the  divergent  views  of  Schaudinn  and  of 
Hartmann  in  studying  the  same  slides.  Greater  certainty  would 
doubtless  be  derived  from  the  study  of  artificial  cultures  if  such 
could  be  made  available. 

Numerous  cultures  of  amebae  have  been  obtained  from  the 
stools  of  cases  of  dysentery,  and  some  from  the  pus  of  amebic 
abscesses  of  the  liver,  the  growth  taking  place  on  agar  in  the  pres- 
ence of  a  single  species  of  bacteria.  With  these  cultures  it  has 
been  possible  to  cause  enteritis  in  monkeys.  Such  cultures  have 
also  been  grown  at  37°  C.  by  A.  W.  Williams1  in  pure  culture  on 
agar  streaked  with  brain  substance  and  with  blood,  and  in  these 
cultures  she  finds  that  the  amebae  approach  in  their  structure 
the  typical  entamebae,  not  only  in  nuclear  structure  and  cyst 
formation,  but  also  in  the  utilization  of  red  blood  cells  as  food. 

1  Soc.  Amer.  Bact.,  New  York  Meeting,  Jan.  2,  1913.  Science  1913;  Vol. 
XXXVIII,  p.  451;  Williams,  A.  W.,  and  Calkins,  G.  N.,  Journ.  Med.  Rsch.,  1913, 
Vol.  XXIX,  pp.  43-56. 


RHIZOPODA  407 

Whitmore1  has  carefully  studied  a  number  of  cultures  of  amebae 
obtained  from  cases  of  dysentery,  one  of  them  from  a  liver  abscess, 
and  has  concluded  that  in  every  instance  the  amebae  were  free- 
living  saprophytic  forms  belonging  to  the  genus  Amoeba  and  not 
in  any  case  parasitic  species. 

Other  Rhizopoda. — The  remaining  orders  of  the  Rhizopoda, 
namely  Helizoa,  Foraminifer,  Radiolaria  and  Mycetozoa  contain 
no  parasitic  forms  of  great  importance  to  human  pathology. 
Plasmodium  brassica  which  causes  tumors  on  the  roots  of  the 
cauliflower  plant  is  of  some  interest.2 

1  Archiv  f,  Protistenkunde,  1911,  Bd.  XXIII,  S.  71-80;  ibid,  pp.  81-95. 

2  See  Doflein,  Protozoenkunde,  1911,  S.  672-678. 


CHAPTER  XXIX. 


SPOROZOA. 

Cyclospora  Caryolytica. — Schaudinn  in  1902  discovered  this 
organism,  which  lives  as  a  parasite  in  the  nuclei  of  epithelial  cells 
of  the  intestinal  mucosa  in  the  common  mole.  It  is  ingested  in 
the  form  of  spores,  from  which  the  slender  young  sporozoites 
escape  in  the  intestine  and  penetrate  the  nuclei  of  epithelial  cells. 
Here  the  parasite  becomes  rounded  and  enlarges,  becoming 
quickly  differentiated  into  either  the  male  or  female  type.  The 
former  type  of  parasite  has  numerous  refractive  granules  in  its 


f 


«?     T 

*;>  ** 


FIG.  177. — Cyclospora  caryolytica.  A,  Male  cells  within  the  nucleus  of  the  host 
cell.  B  and  C,  Reproduction  by  multiple  division  with  final  rupture  of  the  host 
nucleus  in  (C).  (From  Doflein  after  Schaudinn.} 

cytoplasm,  while  the  female  type  has  a  clear  cytoplasm.  The 
parasites  grow  rapidly  and  segment  after  4  to  8  hours,  the  females 
earlier  than  the  males,  and  the  cells  resulting  from  this  segmenta- 
tion, so-called  merozoites  or  agametes,  penetrate  new  nuclei  and 
go  through  the  same  development.  Four  to  five  days  after  in- 
fection of  the  mole,  the  parasites  suddenly  cease  their  asexual 
multiplication.  The  male  parasites,  microgametocytes,  after 
rapid  multiplication  of  nuclei,  give  rise  to  numerous  microgametes 

408 


SPOROZOA 


409 


provided  with  two  flagella.     The  female  cells,  macrogametocytes, 
enlarge  slowly  and  produce  numerous  yolk-like  granules  in  their 


A 


D 


FIG.  178. — Cyclospora  caryolytica.  A,  Female  cell  (agamete)  within  the  host 
nucleus.  B  and  C,  Multiple  division.  D,  A  free  young  female  agamete.  (From 
Dofldn  after  Schaudinn.) 

cytoplasm.     The  nucleus  undergoes  two  reduction  (maturation) 

divisions,   and  one  daughter  nucleus  remains  while  the   others 

A  BCD 


II 


FIG.  179. — Cyclospora  caryolytica.  A,  Fertilization.  B,  Fertilized  cell.  C,  Fer- 
tilized cell  (oocyst)  with  cyst  wall.  D,  E,  F  and  G,  Division  of  the  cyst  contents  to 
form  two  spores,  each  containing  two  sporozoits.  H,  Escape  of  the  sporozoits. 

(From  Doflein  after  Schaudinn.} 

disintegrate.     Several  microgametes  penetrate  the  matured  macro- 
gamete  and  one  of  them  unites  with  the  nucleus.     A  cyst  wall 


4io 


SPECIFIC   MICRO-ORGANISMS 


forms  about  the  fertilized  cell  and  within  this  the  cell  divides 
into  two  and  later  into  four  embryo  parasites,  which  are  enclosed 
in  pairs  in  two  spores  within  the  cyst.  This  escapes  with  the 
feces  of  the  mole  and  serves  to  infect  a  new  host. 

The  invasion  of  the  epithelium  produces  a  severe  diarrhea  in 
the  mole  often  resulting  in  death.     If  the  animal  survives  for 
five  days,  until  after  the  spores  are  formed,  it  then 
usually  recovers. 

Eimeria  Stiedae  (Coccidium  Cuniculi). — This 
very  common  parasite  of  the  rabbit  was  first  de- 
scribed by  Lindemann  in  1 86  5 .  It  lives  and  grows 
within  the  epithelial  cells  of  the  small  intestine,  of 
the  bile  passages  and  of  the  liver  of  rabbits  suffer- 
ing from  coccidiosis,  and  its  oocysts  are  found  in 
the  intestinal  contents  and  in  the  feces  of  such 
animals.  The  oocyst  is  an  elongated  oval,  vari- 

able  in  width  fr°m  IX   to  28^  and  in  length  from 

24  to  4Qju.     It  contains,  when  fully  developed,  four 

,        ,       ,  .  , 

sporozoits  are  de-    spores,  each  of  which  contains  two  embryo  para- 
sjj-es  or  sporozoits.     These  gain  entrance  to  the 


FIG.  1 80.— Ei- 
meria  steidtz. 
Oocyst  containing 
four  spores,  in 
each  of  which  two 


pyle    is 

low.  ~  (From   Do-   intestine  of  a  new  host  along  with  the  food  and  the 

flein    after    Metz-  ,•      -,•        ,.  -, 

ner^  pancreatic  digestion  makes  an  opening  at  one  end 

where  the  wall  is  exceedingly  thin,  the  micropyle, 
and  through  this  opening  the  wedge-shaped  sporozoits  escape. 
They  penetrate  epithelial  cells,  in  which  the  parasite  becomes 
rounded  and  grows  to  a  diameter  of  20  to  5o/z,  destroying 
the  host  cell.  The  nucleus  divides  many  times  and  after 
it  the  cytoplasm,  so  as  to  form  numerous  spindle-shaped 
young  cells,  merozoits  of  agametes,  which  penetrate  new  epi- 
thelial cells  and  pass  through  the  same  cycle.  This  cycle  of 
asexual  multiplication,  schizogony,  is  repeated  many  times  and 
may  lead  to  extensive  destruction  of  intestinal  mucosa,  of  the 
epithelium  of  the  bile  ducts  and  of  liver  substance.  Some  of 
the  growing  parasites  become  differentiated  into  sexual  elements. 
The  female  cell,  macrogametocyte,  accumulates  numerous  large 


SPOROZOA 


411 


granules  in  its  cytoplasm,  and  when  full-grown  the  chromatin 
of  the  nucleus  is  reduced  by  expulsion  of  the  karyosome.  The~ 
matured  cell,  macrogamete,  is  then  ready  for  union  with  the 
microgamete.  The  growing  cell  destined  to  give  rise  to  the  male 
sexual  elements  attains  a  large  size  and  possesses  a  pale  cytoplasm. 
It  is  called  the  microgametocyte.  Its  nucleus  divides  many 
times,  the  small  nuclei  accumulate  near  the  surface  of  the  cell 
and  each  escapes  with  a  small  portion  of  protoplasm  as  a  slender 
motile  microgamete.  The  penetration  of  one  of  Jthese  into  the 
macrogamete  produces  the  fertilized  oocyst,  which  forms  a  thick 


FIG.  181. — Eimeria  steida.  a,  Young  agamete  (merozoit).  b,  Epithelial  cell 
invaded  by  three  young  agametes.  c,  d  and  e,  Stages  in  the  multiple  division  of  the 
agamete.  /,  Young  macrogametocyte.  g,  Full-grown  macrogametocyte.  (From 
Doflein  after  Hartmann.) 

wall  about  itself  and  escapes  to  the  external  world.  Here,  the 
fertilized  cell  divides  to  form  eight  cells,  sporozoits,  which  are 
enclosed  within  four  oval  spores  (two  in  each)  within  the  wall  of 
the  oocyst.  If  this  cyst  is  ingested  by  another  rabbit  the  cycle 
of  development  starts  anew. 

Coccidiosis  is  a  very  common  disease  in  rabbits.  The  animal 
suffers  from  severe  diarrhea  and  loss  of  appetite,  and  becomes 
emaciated.  Young  rabbits  often  die  of  the  disease.  Diagnosis 
is  readily  made  by  finding  the  oocysts  in  the  feces.  Children 
have  been  found  to  be  infected  with  this  organism.  Cattle, 


412  SPECIFIC   MICRO-ORGANISMS 

horses,  sheep  and  swine  are  also  susceptible  and  serious  epizootics 
of  coccidiosis  due  to  E.  stiedce  have  been  observed  in  cattle. 

Eimeria  (Coccidium)  Schubergi. — This  coccidium  occurs  in 
the  intestine  of  a  common  myriapod  (thousand-legged  worm). 
Lithobiusforficatus.  It  is  the  organism  in  which  Schaudinn  worked 
out  the  life-cycle  now  regarded  as  typical  for  Eimeriadse,  and 
which  corresponds  very  closely  to  that  of  E.  stiedce.  (See  Fig. 
78,  page  156). 

Haemoproteus  Columbae. — Celli  and  Sanfelice  in  1891  observed 
this  organism  in  the  red  blood  cells  of  doves.  It  is  widely  dis- 
tributed as  a  parasite  of  wild  doves  and  has  been  found  in  Europe 
and  in  North  and  South  America.  The  life-history  of  the  parasite 
in  the  vertebrate  host  and  its  mode  of  transmission  by  flies  of  the 
genus  Lynchia  has  been  most  fully  studied  by  Aragao.1  In  the 
circulating  blood  of  doves  the  organism  is  most  commonly  seen 
as  a  large  crescent-shaped  structure  occupying  most  of  the  interior 
of  an  erythrocyte  and  crowding  the  nucleus  of  the  latter  to  one 
side  or  encircling  it.  The  outline  of  the  erythrocyte  and  the  out- 
line of  its  nucleus  are  not  distorted.  The  parasites  are  definitely 
recognizable  as  females  and  males,  macrogametocytes  with  granu- 
lar, deeply  staining  cytoplasm  and  microgametocytes  with  a 
paler  cytoplasm.  When  these  are  ingested  by  the  fly  along  with 
its  blood  meal,  the  gametes  arise,  fertilization  takes  place  and 
there  is  produced  a  creeping  ookinete  which  apparently  does  not 
penetrate  the  intestinal  wall  in  the  fly  or  indeed  undergo  any 
further  development  there.  It  gains  the  blood  stream  of  a  new 
host,  especially  young  nestlings,  when  the  fly  bites  them.  It  is 
taken  up  by  a  leukocyte  which  comes  to  rest  in  the  pulmonary 
capillaries  of  the  young  bird.  Here  the  parasite  produces  a  very 
large  cyst  and  divides  to  form  very  numerous  minute  sporozoits. 
When  the  cyst  bursts  these  sporozoits  gain  the  blood  stream, 
penetrate  erythrocytes  and  grow  to  produce  the  gametocytes 
again.  The  asexual  cycle  of  schizogony  seems  to  be  lacking. 

This  organism  is  important  as  a  typical  example  of  Hcemo- 

1  Archi.  /.  Protistenkunde,  1908,  Bd.  XII,  S.  154-167. 


SPOROZOA 


413 


FIG.  182. — H&moproteus  columba.  la  to  3<z,  Development  of  the  female  para- 
site in  the  blood  of  the  dove;  ib  to  36,  development  of  the  male  parasite  in  the  blood 
of  the  dove;  40,  46,  50,  56,  6  to  12,  development  in  the  digestive  tube  of  the  fly 
(Lynchia);  13  to  20,  development  of  the  parasite  inside  leukocytes  in  the  lung  of 
the  dove.  (After  Aragao.) 


414  SPECIFIC   MICRO-ORGANISMS 

proteus,  as  it  is  the  one  species  of  this  genus  in  which  the  life  cycle 
has  been  most  completely  studied. 

HaBmoproteus  (Halteridium)  Danilewskyi. — Grassi  and 
Feletti1  first  clearly  recognized  this  organism  as  a  definite  malarial 
parasite  of  birds.  It  is  widely  distributed  and  has  been  found  in 
very  many  different  birds,  including  sparrows,  doves,  owls,  robins, 
blackbirds  and  crows.  The  life  history  is  imcompletely  known. 
In  the  blood  of  the  infected  bird  the  organism  first  appears  as  a 
small  oval  or  lance-shaped  body  within  the  cytoplasm  of  an  ery- 
throcyte.  This  enlarges,  without  distorting  the  outline  or  dis- 
placing the  nucleus  of  the  blood-cell,  and  stretches  along  one  side 
of  the  cell.  It  curves  about  the  nucleus  and  is  enlarged  at  either 
end  when  fully  developed.  Two  types,  macrogametocytes  and 
A  B  c 


FIG.  183. — Hamoproteus  danilewskyi.  A  and  B,  Fresh  triple  infection  of  red 
blood  cells.  C,  D  and  E,  Growing  parasites,  the  last  two  showing  vesicular  nuclei. 
jF,  Full-grown  halteridium  with  two  nuclei.  (After  Doflein.} 

microgametocytes,  are  easily  recognizable  in  stained  prepara- 
tions. If  blood  containing  these  mature  halteridia  is  diluted 
with  citrated  salt  solution  and  studied  under  the  microscope  the 
further  changes  in  the  sexual  cells  may  often  be  followed.  Each 
gametocyte  bursts  the  erythrocyte  enclosing  it  and  assumes  a 
rounded  outline.  In  the  microgametocyte  the  protoplasmic 
granules  exhibits  violent  agitation  and  several  fine  filamentous 
processes  suddenly  shoot  out  from  its  periphery  and  lash  about. 
After  a  few  moments  these  microgametes  separate  completely 
and  rapidly  swim  away.  Meanwhile,  the  macrogametocyte  has 
escaped  from  its  erythrocyte  and  come  to  rest  in  a  rounded  condi- 
tion. A  microgamete  approaches  and  penetrates  the  macro- 
gamete,  and  in  a  few  minutes  this  fertilized  sphere  elongates  into 

1  Cenlralbl.f.  Bakt.  1891,  Bd.  IX,  S.  403-409;  429-433;  460-467. 


SPOROZOA 


415 


a  curved  spindle  and  actively  creeps  over  the 
slide.  It  is  then  known  as  the  ookinete.  Fur- 
ther development  has  not  been  observed,  but  there 
can  be  little  doubt  that  the  further  stages  of 
sporogony  and  also  the  unobserved  stages  of 
schizogony  in  the  bird  are  somewhat  analogous 
to  those  of  H.  columbcE  or  to  those  of  the  plas- 
modia  of  human  malaria.  Whether  the  halteridia 
which  occur  in  various  species  of  birds  are  all  of 
one  species  cannot  be  decided  without  further  in- 
vestigations. 

Haemoproteus  (Leukocytozoon)  Ziemanni.— 
This  organism  was  doubtless  seen  by  Danilewsky 
in  iSgo.1  Ziemann  in  1898  described  it  as  a  para- 
site in  the  blood  of  hawks.  Its  known  life  history 
is  very  incomplete,  and  even  the  nature  of  the 
blood  cell  containing  it  is  somewhat  doubtful. 
The  youngest  stage  observed  in  the  blood  is  a 


FIG.  184.— 
H  &mo prote  u s 
(Leukocytozoon) 
ziemaunni.  Macro- 
gametocy te  and 
microgametocy  t  e 
(paler.)  (From 
Doflein  after 
Schaudinn.) 


A  B 

FIG.  185. — Hamoproteus  (Leukocytozoon^  ziemanni.  A,  Formation  of  micro- 
gametes  from  the  microgametocy  te;  B,  Fertilization  of  the  macrogamete  by  one  of 
the  microgametes  swarming  about  it.  (From  Doflein  after  Schaudinn.) 

1  Cenlrabl.f.  Bakt.,  1891,  Bd.  IX,  S.  401,  Fig.  i. 


416 


SPECIFIC  MICRO-ORGANISMS 


small  oval  parasite1  situated  at  the  side  of  the  nucleus  of 
the  blood  cell.  The  latter  appears  to  be  an  erythroblast,  an 
immature  red  blood  cell  in  which  there  is  little  or  no  hemoglo- 


D 


FIG.  i 86. — Hamoproieus  (Leukocytozoon)  ziemanni  in  the  blood  of  an  owl  with 
a  pure  infection.  A ,  Young  parasite  in  an  erythroblast.  B,  Growing  parasite  distort- 
ing the  nucleus  of  the  host-cell.  C  and  D,  Further  stages  of  growth  with  marked 
distortion  of  the  nucleus  and  of  the  outline  of  the  host  cell.  E,  Full-grown  macro- 
gametocyte.  F,  Macrogametocyte  and  microgametocyte  in  the  same  field.  G,  Forma- 
tion of  microgametes  from  the  microgametocyte.  (After  micro  photo  graphs  of  Prof. 
F.  G.  Novy.) 

bin.     As  the  parasite  enlarges,  the  host  cell  becomes  swollen  and 
its  nucleus  much  flattened  and  distorted.     The  parasite  itself 

1  The  description  here  given  is  derived  in  part  from  unpublished  observations 
by  Novy  and  MacNeal.  See  Proc.  Soc.  Exp.  Biol.  and  Med.,  1904-05,  Vol.  II, 
pp.  23-28;  American  Medicine,  1904,  Vol.  VIII,  pp.  932-934. 


SPOROZOA 


417 


grows  long  and  rather  slender  and  is  differentiated  to  form  either 
the  male  or  the  female  gametocyte,  readily  distinguished  by 
appearance  in  stained  preparations.  Meanwhile,  the  host  cell 
becomes  very  much  elongated  and  pointed  at  the  ends.  The 
explanation  of  this  peculiar  distortion  of  the  cell  is  unknown,  but 
it  may  be  due  to  the  mechanical  streaming  of  the  blood  acting 
upon  the  bladder-like  cell  which  has  been  deprived  of  elasticity 


FIG  187. — Diagram  of  the  developmental  cycle  of  Proteosoma.  i,  Sporozoit 
entering  an  erythrocyte;  i.  2,  3  and  4,  the  cycle  of  schizogony;  5,  macrogameto- 
cyte;  5^,  microgametocyte;  6,  macrogamete;  6a,  formation  of  microgametes;  7, 
fertilization;  8,  ookinete;  9,  formation  of  sporoblasts  (in  mosquito);  10,  forma- 
tion of  sporozoits;  n.  sporozoit.  (From  Doflein  after  Schaudinn,} 

by  the  destructive  action  of  the  parasite.  The  further  stages  in 
the  cycle  of  sporogony  are  unknown.  An  asexual  multiplica- 
tion probably  occurs  in  some  internal  organs  of  the  bird.  Fan- 
tham  has  observed  schizogony  in  the  spleen  of  Lagopus  scoticus, 
the  red-game  grouse  of  Scotland,  infected  with  a  similar  parasite 
Leukocytozoon  lovati. 

Proteosoma  (Plasmodium)  Praecox.— Grassi  and  Feletti  de- 
scribed this  malarial  parasite  of  birds  and  designated  it  as  Hcem- 
27 


4i8 


SPECIFIC  MICRO-ORGANISMS 


amceba  prcecox.1  The  parasite  is  very  common  in  the  blood  of 
small  birds,  such  as  sparrows,  robins  and  larks,  in  all  parts  of  the 
world.  The  cycle  of  schizogony  is  completed  in  the  peripheral 
circulation.  The  small  merozoit  or  agamete  enters  an  erythro- 
cyte  and  enlarges,  retaining  its  oval  or  circular  form.  The  nucleus 


FIG.  188. — Proteosoma  prcecox  in  the  blood  of  a  field  lark  (Glauda  arvensis). 
A,  Young  parasite  in  a  blood  cell.  B,  Half-grown  parasite  which  has  pushed  aside 
the  nucleus  of  the  erythrocyte.  C.  Parasite  with  clump  of  pigment  and  many  nuclei 
The  nucleus  of  the  erythrocyte  has  been  lost  (uncommon).  D,  Divisicn  into  eighteen 
merozoits.  (From  Doflein  after  Wasielewski.) 

of  the  host  cell  is  pushed  out  of  position  but  its  form  is  not  ma- 
terially altered.  The  full-grown  parasite  segments,  producing 
i o  to  30  merozoits  and  leaving  behind  a  small  residual  body  con- 
taining the  accumulated  pigment,  thus  completing  the  asexual 
cycle,  which  may  be  repeated  many  times.  After  a  time  some 


FIG.  189. — Midgut  of  a  culex  mosquito,  covered  with  oocysts  of  Proteosoma  prcecox 
V,  Vasa  malpighii.     (From  Doflein  after  Ross.) 

of  the  growing  parasites  become  differentiated  to  form  macro- 
gametocytes  and  microgametocytes,  which  are  kidney-shaped  and 
do  not  divide  nor  undergo  further  development  in  the  vertebrate 
host.  When  the  blood  is  drawn  and  diluted  with  citrated  salt  so- 
lution, or  taken  in  by  a  mosquito,  four  to  eight  microgametes  are 

lCentrabl.f.  Bakt.,  1891,  Bd.  IX,  S.  407. 


SPOROZOA 


419 


formed  just  as  has  been  described  for  H.  columbce.  They  are  very 
slender  actively  motile  spindles  without  flagella.  Fertilization 
of  the  macrogamete  and  the  production  of  an  ookinete  takes 
place  in  the  usual  manner.  The  latter  penetrates  the  intestinal 
epithelium  of  the  mosquito  (Culex  sp.)  and  enlarges  to  produce 
a  spherical  cyst  filled  with  an  enormous  number  of  thread-like 
sporozoits.  These  escape  into  the  body  ca- 
vity of  the  mosquito  as  the  cyst  bursts,  and 
are  generally  distributed  throughout  the 
body  of  the  insect.  They  assemble,  prob- 
ably as  a  result  of  some  chemical  stimulus, 
in  the  salivary  glands  of  the  mosquito, 
whence  they  are  injected  into  the  wound  as 
the  insect  bites,  and  at  once  invade  erythro- 
cytes  to  begin  the  cycle  of  schizogony. 

The  discovery  of  the  sexual  cycle  of  pro- 
teosoma  in  the  mosquito  and  the  conclusive 
proof  that  this  form  of  bird  malaria  is  trans- 
mitted by  a  mosquito  stands  to  the  ever- 
lasting credit  of  Ronald  Ross.  His  brillant 
discovery  made  in  India  in  1898,  pointed  FlG-  X9°- — Oocyst  of 

.  .  Proteosoma    prcscox,   de- 

the  way  to  the  Solution   of    the   whole   prob-    veloped  on;  the  intestine 

of  A'edes  (Stegomyia)  ca- 
lopus,  showing  numerous 
sporozoits.  (From  Do- 


flein  after  Neumann.} 


lem  of  the  transmission  of  the  malarial  dis- 
eases and  their  practical  restriction. 

Proteosoma  is  a  favorable  parasite  for 
class  study,  as  it  is  readily  transmitted  from  bird  to  bird  (spar- 
rows or  canaries)  by  injection  of  infected  blood,  and  the  para- 
sites often  become  very  numerous  in  the  blood.  There  seems 
to  be  no  good  reason  for  placing  this  organism  in  a  separate  genus 
from  the  human  malarial  parasites. 

Plasmodium  Falciparum  (Praecox). — Laveran  in  1880  dis- 
covered the  first  malarial  parasite  in  the  blood  of  man  and  cor- 
rectly interpreted  his  observations.  The  distinctions  between  the 
three  species  was  recognized  by  Golgi,  and  the  life  history  of  the 
parasites  and  especially  their  relation  to  mosquitoes  and  insects 


420 


SPECIFIC   MICRO-ORGANISMS 


in  general  has  been  most  thoroughly  studied  by  Grassi.1   PL  falcip- 
arum  is  the  parasite  of  estivo-autumnal  or  pernicious  malaria  of 


FIG.  191. — Plasmodium  falciparium,  forms  in  the  asexual  cycle  (schizogony). 
A,  Multiple  infection  of  an  erythrocyte,  showing  signet  rings  and  parasites  attached 
to  the  external  surface.  B  and  C,  Growing  parasites  with  Mauer's  granules  in  the 
erythrocytes.  D,  Growing  parasite  without  granulation  of  the  hemoglobin.  E,  Half- 
grown  parasite  showing  pigment.  F,  and  G,  Multiple  division  (sporulation),  rarely 
seen  in  the  peripheral  blood.  (After  Doflein.} 

man.     The  young  organism  is  i  to  1.5/4  in  diameter.     It  pene- 
trates a  red  blood  cell  and  enlarges.     A  vacuole  appears  in  the 

center,  giving  the  parasite  the 
appearance  of  a  signet  ring,  the 
setting  being  represented  by 
the  nucleus  or  chromatin  gran- 
ule which  stains  violet  red  with 
the  Romanowsky  stains.  The 
parasite  attains  a  diameter  of 
about  6/j,  when  it  segments  to 
produce  7  to  16  merozoits  or 
agametes  which  enter  new  ery- 


FIG.  192. — Section  through  a  capil- 
lary in  the  brain,  showing  numerous  di- 
viding forms  of  the  non-pigmented  type 
of  PL  falciparum.  (Stained  prepara- 
tion.) From  Doflein  after  Mannaberg.) 


throcytes  and  repeat  the  cycle. 
The  larger  stages  of  this  cycle  of  schizogony  are  rarely  seen 
in  the  peripheral  circulation,  and  the  segmentation  of  the 

1  Grassi:  Die  Malaria,  lite  Auflage,  Jena,  1901. 


SPOROZOA 


421 


parasite  occurs  in  the  capillaries  of  the  internal  organs.  The 
cycle  probably  requires  48  hours  for  its  completion.  The  ery- 
throcyte  is  not  enlarged  by  the  growth  of  the  parasite  within 


FIG.  193. — Plasmodium  falciparum.    Stages  in  the  development  of  the  gametocytes 
(crescents).     X22oo.     (After  Doflein.) 

it,  but  tends  rather  to  become  smaller.     Maurer  has  observed  an 
irregular  granulation  of  the  erythrocytes.     Why  the  cells  con- 


i  .  .... 

F     em    e      c  I  F          em     e      c    I 

FIG.  194. — Sections  through  the  stomach  wall  of  Anopheles  showing  stages  in 
the  development  of  PI.  falciparum.  A,  Fixed  a  few  hours  after  the  infective  feeding, 
showing  ookinetes  within  the  lumen  and  two  in  the  cuticula  of  the  epithelium.  5, 
Fixed  a  few  days  after  the  infective  feeding,  showing  the  partly  grown  oocyst  in  the 
stomach  wall.  F,  Fat  surrounding  the  stomach;  em,  tunica  elastico-muscularis;  e, 
epithelium;  c,  cuticula;  I,  lumen  of  stomach. 

taining  the  larger  forms  should  remain  in  the  internal  capillaries 
of  the  body  is  not  definitely  known. 

The  gametocytes  develop  by  the  growth  of  ordinary  merozoits, 


422 


SPECIFIC   MICRO-ORGANISMS 


marrow  and 


which  become  crescentic  early  in  their  development  and  differen- 
tiated into  deeply  staining  macrogametocytes  and  pale-staining 
microgametocytes.  These  are  produced  especially  in  the  bone 
circulate  in  the  peripheral  blood.  Further 
development  takes  place  when  the  blood 
is  taken  into  the  stomach  of  a  mosquito 
of  the  genus  Anopheles.  Here  the  mi- 
crogametes,  slender  actively  motile 
threads,  are  given  off  by  the  microgam- 
etocyte  and  fertilize  the  macrogam- 
etes,  producing  ookinetes  which  ac- 
tively penetrate  the  epithelium.  In  the 
wall  of  the  mosquito's  stomach  each 
ookinete  gives  rise  to  a  rapidly  growing 


FIG.  195. — Digestive  tract 
of  Anopheles,  the  stomach  of 
which  is  covered  with  numer- 
ous oocysts  of  PL  falciparum, 
-viewed  from  the  left  side,  c, 
Cloaca;s,  stomach;  o,  oocysts 
of  Plasmodium;  mt,  malpigh- 
ian  tubules;  sb,  sucking  blad- 
ders; sg,  salivary  gland. 
(From  Doflein,  modified  after 
Ross  and  Grassi.) 


FIG.  196. — Plasmodium  falciparum. 
Ripe  sporozoits  arranged  about  residual 
bodies  within  the  oocyst,  cut  in  various 
directions  (7  to  8  days  after  infection  of 
the  mosquito).  (From  Doflein  after 
Grassi.} 


SPOROZOA 


423 


cyst  and  within  this  an  enormous  number  of  very  slender  sporozoits 
are  developed.  The  ripe  cyst  bursts  into  the  body  cavity  and  the- 
sporozoits  become  generally  distributed  throughout  the  body  of 
the  insect  and  later  assemble  in  the  secreting  cells  of  the  salivary 
glands,  from  which  they  escape  into  the  human  host  when  the 
mosquito  bites.  The  cycle  in  Anopheles  requires  eight  days  at  a 
temperature  of  28°  to  30°  C.  At  temperatures  below  iy°C.  the 
microgametes  are  not  produced. 

Development   of   the   estivo-autumnal  parasite   through   the 


\ 


FIG.  197. — Section  through  salivary  gland  of  Anopheles  showing  numerous 
sporozoits  of  Plasmodium  falciparum.  i,  Fat  bodies;  2,  gland  duct;  3,  sporozoits  of 
Plasmodium;  4,  Secretion  in  the  gland  cells.  (From  Doflein  after  Grassi.} 

stages  of  schizogony  has  been  obtained  by  Bass  and  Johns1  in  the 
test-tube,  in  a  medium  consisting  of  defibrinated  blood  to  which 
0.5  per  cent  glucose  has  been  added.  They  were  able  to  keep  the 
organisms  alive  for  ten  days  at  a  temperature  of  40°  C.,  during 
which  period  the  developmental  cycle  was  repeated  four  or  five 
times.  Their  findings  have  been  confirmed  by  other  investi- 
gators. More  recently  Joukoff 2  has  reported  partial  development 
in  the  test-tube,  of  the  cycle  of  sporogony  in  the  case  of  PL  falci- 

1  Journ.  Exp.  Med.,  1912,  Vol.  XVI,  pp.  567-579. 

2  Compt.  Rend.  Soc.  BioL,  1913,  Vol.  LXXIV,  pp.Ji36-i38. 


424 


SPECIFIC   MICRO-ORGANISMS 


parum,  and  greater  success  with  PL  malaria.     Details  of  this  work 
have  not  yet  been  published. 

Plasmodium  Vivax. — The  parasite  of  tertian  malaria  is  dis- 
tinctly different  from  the  estivo-autumnal  parasite.     The  young 


FIG.  198. — Plasmodium  vivax.  Stages  of  growth  in  the  asexual  cycle,  commonly 
seen  in  the  peripheral  blood.  Three  of  the  cells  show  granules  in  the  hemoglobin, 
the  stippling  of  Schiiffner.  X22oo.  (After  Do flein.} 

merozoit  is  i  to  2/4  in  diameter  and  practically  not  to  be  distin- 
guished, but  very  early  in  its  growth  it  becomes  actively  ameboid 
and  extends  irregular  and  slender  processes  into  the  protoplasm 
of  its  host  cell.  As  the  parasite  enlarges,  the  erythrocyte,  often 
but  not  always,  becomes  swollen,  paler,  and  shows  a  coarse  granu- 
A  B 


FIG.  199. — Plasmodium  vivax.  Multinu- 
cleated  stage  preceding  division  and  the  stage 
of.  multiple  division  (sporulation) ;  found  in  the 
blood  just  before  and  during  a  chill.  X22oo. 
(After  Do  flein.} 


FIG.  200. — Plasmodium 
vivax.  Double  infection  of 
a  red  blood  cell  which  is 
considerably  enlarged  as  a 
result;  Schuffner's  stippling 
slight.  X22CO.  (After 
Do  flein.} 


lation,  the  stippling  of  Schueffner.  The  parasite  often  attains 
a  diameter  greater  than  that  of  the  average  blood  cell  before  it 
segments.  The  segmentation  gives  rise  to  from  15  to  30  mero- 
zoits  which  enter  new  erythrocytes  and  begin  the  cycle  anew. 
This  complete  cycle  of  schizogony  takes  place  in  the  peripheral 
circulation  and  requires  almost  exactly  48  hours. 


SPOROZOA  425 

The  young  parasites  destined  to  become  gametocytes  ex- 
hibit relatively  less  ameboid  movement.  Their  pigment  exists  as 
large  granules,  some  of  them  even  rod-shaped.  The  macrogame- 
tocyte  attains  a  diameter  of  15  to  25/1  and  usually  destroys  its 
erythrocyte  and  escapes  from  it  entirely.  The  cytoplasm  stains 
deeply  with  methylene  blue.  The  microgametocyte  is  smaller 
with  paler  cytoplasm.  The  development  of  the  parasite  in  the 
mosquito  (Anopheles)  is  wholly  analogous  to  that  of  PL  falciparum, 
although  there  are  some  slight  morphological  differences  ob- 
served. Development  ceases  at  temperatures  below  16°  C. 

ABC  D 


FIG.  201. — Plasmodium  vivax.  Stages  in  growth  of  the  sexual  cells  (gametocytes). 
A  and  B,  Young  sexual  cells  distinguished  from  the  agametes  by  the  absence  of 
vacuoles  and  the  more  regular  outline.  C,  Full-grown  macrogametocyte.  D,  Full- 
grown  microgametocyte.  X22oo.  (After  Doflein.} 

Plasmodium  Malariae. — The  young  quartan  parasite  is  not 
characteristic,  but  in  its  growth  it  soon  stretches  as  a  band  across 
the  erythrocyte.  Later  it  almost  fills  the  cell  and  then  segments, 
producing  6  to  14,  most  often  8,  merozoits.  The  infected  erythro- 
cyte is  not  enlarged  or  distorted  nor  does  it  become  pale  or  show 
granulation.  The  gametocytes,  when  stained,  are  not  very 
different  in  appearance  from  the  asexual  cells.  In  the  living 
preparation  they  show  much  more  active  protoplasmic  move- 
ment. The  sexual  cycle  takes  place  in  Anopheles  and  agrees  very 
well  with  that  of  the  other  two  malarial  parasites,  as  far  as  it  has 
been  studied. 

Malaria  is  probably  the  most  important  as  well  as  the  most 
well-known  human  disease  due  to  protozoa.  It  is  characterized 


426 


SPECIFIC  MICRO-ORGANISMS 


by  recurrent  paroxysms  of  fever  with  afebrile  intervals,  progress- 
ive anemia  and  weakness,  with  the  accumulation  of  a  dark  brown 
or  black  pigment  in  the  spleen  and  liver.  This  pigment  is  pro- 
duced by  the  parasites  and  set  free  into  the  blood  when  they 


FIG.  202. — Plasmodium  malaria.  Stages  of  the  asexual  cycle  in  the  circulating 
blood.  Note  the  absence  of  granulation  from  the  hemoglobin  and  the  uniform  size 
of  the  red  blood  cells.  X22oo.  (After  Do flein.) 

segment.  The  estivo-autumnal  malaria  caused  by  PL  falci- 
parum  shows  a  somewhat  irregular  and  not  very  characteristic 
fever  curve,  but  usually  there  is  fever  every  day  (quotidian  fe- 
ver). The  tertian  fever  due  to  infection  with  PL  vivax  is  char- 


FIG.  203. — Plasmodium  malaria.  Sexual  cells  in  the  circulating  blood.  A ,  Young 
gametocyte.  B,  Full-grown  macrogametocyte.  C,  Full-grown  microgametocyte. 
X  2  200.  (After  Do  flein.} 

acterized  by  febrile  attacks  recurring  at  intervals  of  48  hours  and 
bearing  a  very  definite  relation  to  the  asexual  cycle  of  the  para- 
site. The  segmentation  of  the  plasmodium  is  coincident  with 
the  chill  and  the  rise  in  the  patient's  temperature.  In  quartan 


SPOROZOA  427 

malaria  due  to  infection  with  PL  malaria,  the  fever  recurs  at 
intervals  of  72  hours,  again  at  the  stage  of  segmentation  in  die- 
asexual  cycle  of  the  parasite.     Obviously  an  association  of  two 
or  more  crops  of  parasites  reaching  maturity  at  different  times 
may  give  rise  to  a  variety  of  fever  curves. 

The  diagnosis  of  malaria  is  most  conclusively  established  by 
recognizing  the  parasites  in  the  blood  of  the  patient.  One  should 
examine  a  fresh  drop  of  blood,  unstained,  under  the  microscope, 
and  also  thin  films  of  blood  stained  with  some  one  of  the  Ro- 
manowsky  stains.  The  parasites  may  be  very  scarce  in  old  cases 
and  especially  in  those  patients  who  have  been  treated. 

The  mosquitoes  which  transmit  human  malaria  were  first 
recognized  by  Ross  and  have  been  most  thoroughly  studied  by 
Grassi.  The  mosquito  is  capable  of  causing  malaria  only  after 
it  has  fed  upon  a  person  harboring  the  parasite  in  his  blood.1  The 
members  of  the  genus  Culex,  the  most  common  mosquitoes,  do 
not  permit  the  development  of  the  plasmodia  within  them,  but 
this  occurs,  so  far  as  is  known,  only  in  certain  species  of  the  genus 
Anopheles,  A.  maculipennis  appears  to  be  the  most  important 
species.  It  is  easily  recognized  by  the  four  small  black  spots  on 
each  wing  due  to  a  relative  accumulation  of  pigmented  scales  in 
these  situations.  The  members  of  the  genus  Anopheles  are  read- 
ily distinguishable  from  Culex  by  the  form  and  arrangement  of 
their  eggs,  the  form  and  position  of  the  larvae  and  by  the  general 
form  and  structure  of  the  adult  insect,  as  well  as  its  posture  when 
at  rest. 

The  restriction  and  prevention  of  malaria  is  founded  upon  the 
knowledge  of  its  nature  and  its  mode  of  spread.  The  measures 
include  (i)  the  destruction  of  malarial  parasites  in  man  by  thor- 
ough treatment  of  the  disease  with  quinine,  (2)  destruction  of 
mosquitoes  and  mosquito  larvae  and  the  drainage,  oiling  or  screen- 
ing of  their  breeding  places,  and  (3)  exclusion  of  mosquitoes  from 
contact  with  infected  persons  and  also  from  contact  with  healthy 
persons,  by  the  use  of  screens.  The  thorough  application  of 

1  Fermi  and  Lumbau:  Centrbl.  f.  Bakt.,  1912,  Bd.  LXV,  pp.  105-112. 


428 


SPECIFIC  MICRO-ORGANISMS 

Culex  Anopheles 


6 


Q 


FIG.  204. — Comparison  of  Culex  and  Anopheles.  Eggs,  larvae  (note  position), 
position  of  insects  at  rest,  wings,  heads  showing  antennae  and  palpi.  (From  Jordan 
after  Kolle  and  Hetsch.*) 


SPOROZOA  429 

these  measures  has  demonstrated  the  possibility  of  effectively 
controlling  this  disease  even  in  the  tropics. 

Plasmodium  Kochi. — This  is  a  malarial  parasite  which  causes 
a  mild  fever  in  monkeys.  It  is  not  transmissible  to  man.  Other 
species  of  malarial  parasites  have  been  recognized  in  these 
animals. 

Babesia1  Bigemina. — Smith  and  Kilborne  discovered  this 
organism  in  the  red  blood-corpuscles  of  cattle  suffering  from 
Texas  fever.  The  parasite  is  pear-shaped,  2  to  4/x  long  and  1.5  to 
2/x  wide  and  usually  occurs  in  pairs  within  the  erythrocytes.  The 


J          I     *      /         * 


,/ 


'* 


FIG.  205. — Babesia  bigemina.     Characteristic  forms  in  the  peripheral  blood  of  cattle. 
X2000.     (After  Doflein.) 

cytoplasm  is  quite  clear  without  granules  or  pigment  and  contains 
one  or  two  chromatin  bodies.  Minute  ameboid  forms  are  also 
found.  Multiplication  apparently  takes  place  by  longitudinal 
division  of  the  pear-shaped  forms  as  well  as  by  multiple  division 
of  the  ameboid  forms.  Macrogametocytes  and  microgametocytes 
have  been  recognized.  The  transmission  of  the  parasite  from 
animal  to  animal  is  effected  by  the  cattle  tick,  Boophilus  boms, 
(Rhipicephalus  annulatus)  as  was  conclusively  demonstrated  by 
Smith  and  Kilborne,  the  first  instance  in  which  such  a  relation 

1  The  generic  name  Pyrosoma  bestowed  by  Smith  and  Kilborne  in  1893  is  incor- 
rect, because  this  is  the  name  of  a  genus  of  marine  animals  belonging  to  the  Tuni- 
cata.  Babesia  proposed  by  Starcovici  in  1893  has  the  next  claim  to  priority. 


43°  SPECIFIC   MICRO-ORGANISMS 

was  proved  for  any  blood-sucking  invertebrate.  The  details  of 
the  life  cycle  in  the  tick  are  unknown.  It  is  certain  however 
that  the  infection  is  conveyed  to  the  next  generation  of  ticks 
through  the  eggs  and  that  these  young  ticks  are  capable  of  in- 
fecting cattle.  Renewed  investigation  of  the  parasite  is  much  to 
be  desired. 

Texas  fever  is  a  very  important  disease  of  cattle  in  the  southern 
United  States  and  a  similar  disease  occurs  in  Europe,  Africa  and 
South  America.  Young  cattle  usually  survive  the  disease  and 
become  immune.  Older  cattle  imported  into  the  endemic  area 
contract  Texas  fever  and  usually  die  of  it.  Immunity  may  be 
conferred  by  injecting  blood  which  contains  a  small  number  of 
parasites,  taken  from  an  animal  which  has  passed  the  acute  stage 
of  the  disease.  Restriction  of  the  Texas-fever  area  in  the  United 
States  is  slowly  progressing  as  a  result  of  systematic  eradication 
of  the  tick. 

Babesia  Canis.— This  organism  occurs  in  the  blood  of  dogs 
suffering  from  the  so-called  malignant  jaundice,  and  has  been 
carefully  studied  by  modern  methods  by  Nuttall  and  Graham- 
Smith  and  later  by  Breinl1  and  Hindle.  In  morphology  and  life 
history  it  agrees  with  B.  bigemina  as  far  as  these  have  been  worked 
out,  but  B.  canis  is  incapable  of  infecting  cattle.  The  infection 
is  transmitted  to  dogs  by  several  different  species  of  ticks. 

Gregarina  Blattarum. — This  organism  lives  as  a  parasite  in 
the  intestine  of  the  common  cockroach  Periplaneta  orientalis,  and 
is  therefore  liable  to  be  found  in  human  food,  and  at  times  in 
specimens  from  human  cases  submitted  to  microscopic  study, 
probably  because  of  accidental  presence  of  cockroaches  in  the 
containers  employed.  The  vegetative  cells  are  elongated,  often 
attached  together.  The  spore  cyst  results  from  the  union  of  two 
cells  and  the  subsequent  repeated  division  of  the  fertilized  cell 
to  produce  an  enormous  number  of  spores.  These  spores  are 
discharged  from  the  cyst  when  it  enters  a  fluid  medium.  When 
fully  developed,  each  spore  contains  eight  sporozoits. 

1  Ann.  Trap.  Med.  and  Parasitol.,  Vol.  II.,  pp.  233-248. 


SPOROZOA 


431 


Nosema  Bombycis. — This  organism  was  discovered  by  Naegeli 
in  1857.  It  is  an  example  of  the  Neosporidia  and  is  of  peculiar 
interest  as  the  cause  of  pebrine,  the  disease  of  silkworms  studied 
by  Pasteur  in  1866-1870,  and  largely  eradicated  by  application 
of  the  methods  devised  by  him  as  a  result  of  his  investigations. 


FIG.  206. — Gregarina  blattarium.  I,  Two  individuals  stuck  together.  //,  Cysts 
with  conjugated  cells  and  developing  spores.  Ill  A,  Unripe  spore  with  undivided 
contents.  IIIB,  Ripe  spore  with  eight  sporozoits;  ek,  ectoplasm;  en,  endoplasm; 
cu,  cuticula;  pm,  protomerit;  dm,  deuteromerit;  n,  nucleus;  pn,  spores;  rk,  residual 
body;  sk,  sporozoits.  (From  Doflein  after  R.  Hertwig.) 

The  spore  of  N.  bombycis  is  1.5  to  2/z  wide  by  3/1  long.  If  treated 
with  nitric  acid  it  swells  and  reaches  a  length  of  6/*  and  extends 
a  slender  thread  which  may  be  lo/x  long.  The  spore  is  ingested 
by  the  silkworm  and  in  its  intestine  the  ameboid  parasite  es- 
capes and  penetrates  the  epithelium.  It  may  pass  to  any  part 
of  the  host  to  undergo  its  further  development.  Multiplication 


432 


SPECIFIC   MICRO-ORGANISMS 


of  the  small  rounded  agamete  results  in  the  formation  of  long 
chains  of  oval  bodies  inside  a  cell  of  the  host.  From  these  the 
spores  are  again  produced.  Pebrine  is  a  disease  of  the  greatest 


FIG.  207. — Nosema  bombycis.  Section  of  intestinal  epithelium  of  silkworm 
showing  spores  of  Nosema  and  also  the  peculiar  multiplication  resembling  the 
growth  of  a  mold.  (From  Doflein  after  Stempell.}  (See  also  Fig.  81,  p.  159.) 

importance  to  the  silkworm  industry.  It  is  effectively  restricted 
by  a  careful  microscopic  examination  of  all  the  silkworm  eggs 
and  the  exclusion  and  destruction  of  all  those  in  which  the  para- 
site exists  (Pasteur's  method). 


CHAPTER  XXX. 


CILOPHORA. 

Paramaecium  Caudatum. — This  is  the  most  common  infusor- 
ian  met  with  in  stagnant  water.  Its 
length  varies  from  120  to  325^1.  The 
cell  is  spindle-shaped  with  a  deep  oral 
groove  which  takes  a  spiral  course  on 
one  side  of  the  body.  The  surface  is 
thickly  set  with  active  cilia.  Food  par- 
ticles are  swept  into  the  oral  groove, 
enter  the  cytoplasm  at  its  bottom  and 
circulate  in  the  cell  within  food  vac- 
uoles.  Near  the  center  of  the  cell  is 
a  large  macronucleus  and  near  it  a 
smaller  micronucleus.  Multiplication 
takes  place  by  simple  longitudinal  or 
oblique  division. 

Conjugation  is  isogamic.  The  simi- 
lar conjugating  cells  adhere  to  each 
other,  the  micronuclei  divide  twice  and 
three  of  the  four  nuclei  thus  produced 
disintegrate,  as  does  also  the  macro- 
nucleus.  The  remaining  micronucleus 
divides  into  two  and  one  of  these  passes 
into  the  other  conjugating  cell  in  ex- 
change for  a  similar  element.  The 
newly  acquired  element  unites  with  the 
element  which  remained  behind  to  form 
the  new  nucleus.  The  new  nucleus  di- 
vides three  times  in  succession  to  form 
28  433 


FIG.  208. — Paramcecium 
caudatum.  K,  Macronucleus; 
NK,  micronucleus;  C,  gullet; 
N,  food  vacuoles;  CV,  contrac- 
tile vacuoles.  (After  Doflein.} 


434 


SPECIFIC   MICRO-ORGANISMS 


eight  nuclei,  of  which  four  enlarge  to  become  macronuclei,  one  re- 
mains as  a  micronucleus  and  three  disintegrate  and  disappear. 
The  one  micronucleus  then  divides  by  mitosis  and  the  cell  divides 
to  form  two  paramaccia,  each  containing  one  micronucleus  and  two 
macronuclei.  The  next  division  gives  rise  to  cells  containing 
A  the  normal  number  of  nuclei,  one  micronucleus  and 

one  macron ucleus. 

The  paramecia  are  large  saprophytic  organisms, 
easily  kept  under  cultivation  in  the  laboratory,  and 
they  have  been  very  extensively  studied.  Many 
conceptions  founded  upon  these 
studies  are  considered  to  have  a 
broad  bearing  upon  the  physiology  of 
all  living  cells.  For  example  Jen- 
nings1 has  found  that  conjugation 
serves  two  purposes,  (i)  to  provide 
chemical  stimulation  of  cell  division 
and  (2)  to  insure  variety  in  the  de- 
scendants. The  variety  in  the  de- 
scendants is  a  result  of  the  exchange 
of  nuclear  material.  Calkins2  has 


FIG.  209. — Paramaecia  drawn  at      ..  .    ..  ... 

the  same  magnification.     A.  Para-     discovered    a    specialization    of   func- 

m&cium   caudatum. 
cium  putrinum. 


B.    ParamcE- 


tion  in  paramecium  in  respect  to  con- 
jugation and  concludes  that  in  some 
of  the  descendants  of  an  ex-conjugant  the  ability  to  conju- 
gate is  in  abeyance,  thus  suggesting  a  resemblance  to  the  somatic 
cells  of  a  metazoon,  while  other  descendants  retain  this  function 
and  are  therefore  analogous  to  the  germ  cells  of  a  metazoon. 

Three  other  species  of  paramecium  are  recognized,  namely, 
P.  aurelia,  P.  bursaria  and  P.  putrinum. 

Opalina  Ranarum. — This  is  a  common  parasite  in  the  in- 
testine (cloaca)  of  the  frog.  It  reaches  a  large  size,  600  to  Sooju 
in  diameter,  is  flattened  and  somewhat  irregular  in  outline.  The 


1  Harvey  Lectures,  1911-12,  pp.  256—276. 

2Proc.  Soc.  Exp.  Biol.  and  Med.,  1913,  Vol.  X,  pp.  65-67. 


CILOPHORA 


435 


ectoplasm  is  striated  and  there  are  very  many  nuclei  in  the  in- 
terior of  the  cell.  In  the  springtime,  as  the  frogs  enter  water  to 
spawn,  the  parasites  divide  rapidly  and  give  rise  to  cysts  20  to  40/1 
in  diameter.  These  escape  into  the  slime  and  are  ingested  by 
the  growing  tadpoles.  In  the  cloaca  the  cells  escape  from  the 
cysts.  They  are  differentiated  into  male  and  female  gametes  and 
fuse  to  form  one  cell  which  grows  and  multiplies  in  the  developing 
frog. 


FIG.  210. — Opalina  ranarum,  showing  the  numerous  vesicular  nuclei.     A,  Ordinary 
form.     B,  Dividing  form.     (From  Doflein  after  Zeller.) 

Balantidium  Coli. — This  parasite  of  the  human  intestine  was 
described  by  Malmsten  in  1857.  Its  normal  habitat  seems  to 
be  in  the  large  intestine  of  swine,  where  it  is  commonly  found  in 
large  numbers.  The  cell  is  a  short  oval,  50  to  yo/z  wide  and 
70  to  IOOM  long,  rarely  larger.  Its  surface  is  covered  with  active 
cilia,  and  there  is  a  short  oral  groove  at  the  anterior  end.  The 
cytoplasm  contains  drops  of  fat  and  food  vacuoles,  often  red 
blood  cells  and  leukocytes  of  the  host.  The  principal  nucleus 
is  kidney-shaped  and  the  accessory  nucleus  lies  in  contact  with 
it.  Multiplication  takes  place  by  simple  transverse  fission. 
Conjugation  and  cyst  formation  have  been  observed. 


436 


SPECIFIC   MICRO-ORGANISMS 


Bal.  coli  is  sometimes  found  in  man  in  cases  of  intestinal  dis- 
order with  diarrhea.     Its  possible  causal  relation  to  the  patho- 
A  B  c  D 


FIG.  211. — Balantidium  coli.  A.  Fully  developed  individual,  showing  the 
nucleus  above  at  the  right  and  a  food  particle  below.  B  and  C,  Division  stages.  D, 
Conjugation.  (From  Doflein  after  Leuckart.} 

logical  condition  is  not  conclusively  ascertained.  In  some  in- 
stances the  cells  of  Balantidium  have  been  found  deeply  situ- 
ated in  inflamed  intestinal  wall.  Brooks1  observed  Bal.  coli  in 


FIG.  212.  —  Section  through  the  intestinal  wall  in  a  case  of  enteritis  due  to 
Balantidium.    S,  Serosa;  M,  Muscularis;  B,  Balantidia.     (From  Doflein  after  Solow- 


several  cases  of  dys'entery  in  Orangoutangs  in  the  New  York 
Zoological  Park  and  Brumpt2  has  been  able  to  transfer  balan- 

^roc.  N.  Y.  Path.  Soc.,  1903,  Vol.  Ill,  pp.  28-39. 

2  Compt.  Rend.  Soc.  Biol.,  1909,  Vol.  LXVII,  pp_.  103-105. 


CILOPHORA  437 

tidium  infection  from  monkey  to  swine  and  back  to  monkey. 
Still  there  is  perhaps  some  question  as  to  the  identity  of  the  para- 
sites found  in  man  and  in  hogs. 

Balantidium  Minutum. — Schaudinn  in  1899  observed  this 
organism  in  the  human  feces.  It  is  smaller  than  Bal.  coli,  the 
greatest  measurements  being  20X30^,  and  the  oral  groove  ex- 


FIG.  213. — Spharophrya  pusilla  within  a  paramaecium.  At  one  place  there  are 
four  parasites"and  a  fifth  is  escaping.  Higher  up,  one  of  the  parasites  is  just  pene- 
trating the  host,  and  a  single  parasite  is  seen  near  the  center  of  the  paramaecium. 
(From  Doflein  after  Butschli.} 

tends  more  than  half  way  back  along  the  side  of  the  cell.  It 
probably  occurs  rarely  in  the  human  small  intestine.  Other 
species  of  balantidium  have  been  described. 

Sphaerophrya  Pusilla. — This  organism  is  of  peculiar  interest 
because  it  lives  as  a  parasite  within  another  protozoon,  the  para- 
mecium.  The  cell  of  Sph.  pusilla  is  spherical,  20  to  40/4  in  diam- 
eter, and  provided  with  sucking  tentacles  and  cilia  when  outside 
the  body  of  the  host. 


INDEX  OF  NAMES 


Abbe,  16 
Abbott,  106 
Adil-Bey,  370 
Agramonte,  369,  370 
Anderson,  F.,  328 
Anderson,  J.  F.,  283,  375 
Appert,  6 
Aragao,  412 
Aristotle,  3 
Arloing,  276 
Armato  (d'Armato),  15 
Arnold,  67 
Arrhenius,  204 
Arustamoff,  249 
Ashburn,  374 
Atkinson,  293 
Audouin,  10,  235 
Axenfeld,  296 

Bacon,  15 

Baeslack,  374 

Bail,  205 

Bang,  341 

Banzhaf,  209,  293 

Bass,  423 

Bassis,  10,  235 

Bastian,  4 

Bataillon,  299 

Bateman,  388 

Becker  274 

Behring    (von    Behring),   12,   208,   281, 

292 

Berg,  238 
Besredka,  225,  330 
Beurmann  (de  Beurmann),  244 
Beyerinck,  14 
Biggs,  290 
Billroth,  ii 
Birt,  374 
Boidin,  274 
Bolduan,  193 
Bellinger,  246,  276 
Bolton,  91,  188,  373 
Bordet,  204,  215,  216,  217,  228 
Breinl,  430 
Brem,  314 
Bretonneau,  289 
Brieger,  203 
Briscoe,  311 


Brooks,  436 
Brown,  L.,  310 
Bruce,  321,  384,  388 
Brumpt,  436 
Buchner,  125,  212,  227 
Bumm,  250 
Burvill-Holmes,  314 
Buschke,  244 
Busse,  244 

Cagniard-Latour,  6 

Calkins,  406,  434, 

Calmette,  309,  315 

Carle,  278 

Carroll,  368,  369 

Castellani,  388 

Celli.  412 

Chace,  102 

Chagas,  392,  394 

Chambers,  384 

Chauveau,  227 

Chevalier,  15 

Citron,  210,  215,  229,  361 

Clark,  H.  W.,  182,  188 

Clegg,  312 

Cohn,  203 

Cohn,  F.,  5 

Cole,  259 

Conor,  375 

Conseil,  375 

Cornet,  37 

Cornevin,  276 

Councilman,  93,  254 

Couret,  312 

Craig,  374 

Cumming,  372 

Danilewsky,  391,  415 
d'Armato,  15 
Davaine,  10,  270,  400 
DeBeurmann,  244 
De  Schweinitz,  373 
Delafield,  61 
Dobell,  353 
Doerr,  374 

Doflein,  151,  378,40? 
Donn6,  10 
Dorset,  94,  373 
Douglas,  217 


439 


440 

Dubard,  299 
Duclaux,  14 
Ducrey,  298 
Durham,  211 
Dusch,  6 

Button,  353,  354,  388 
Duval,  312 

Eberth,  330 

Ehrenberg,  4,  353 

Enrlich,  204,  205,  207,  208,  211,  214,  218, 

227,  289,  294 
Eichorn,  341 
Einhorn,  102 
Elmassian,  388 
Endo,  354 
Erb,  253 
Ermengem  (von  Ermengem),  53,   283, 

284 

Escherich,  324,  326,  327 
Esmarch,  no,  345 
Evans,  387 
Eyre,  322 

Fabyan,  341 

Fantham,  390,  417 

Fehleisen,  260 

Feletti,  414,  417 

Fermi,  427 

Ferran,  222,  351 

Feser,  276 

Finkler,  352 

Fitzgerald,  338 

Flexner,  219,  255,  337,  374,  375 

Flugge,  1 88 

Forde,  388 

Forscher,  278 

Fortineau,  274 

Fracas torius,  9,  201 

Frankel,   *     257 

Freer,  317 

Friedlander,  257,  327 

Frisch  (von  Frisch),  327 

Frosch,  368 

Fuller,  86,  179,  181 

Gaffky,  330 
Gage,  182 
Galen,  9 
Galileo,  15 
Gamaleia,  352 
Garbat,  210,  229,  361 
Garre,  266 
Gartner,  328 
Gatewood,  298 
Geppert,  74,  75 
Gemy,  248 


INDEX  OF  NAMES 


Gengou,  217 
Gessard,  343 
Gibson,  293 
Giemsa,  42 
Gilchrist,  244,  342 
Goldberger,  375 
Golgi,  419 
Goodsir,  267 
Gordon,  16,  322 
Gorsline,  135 
Graham-Smith,  430 
Gram,  44,  59 
Grassi,  414,  417,  420,  427 
Grawitz,  238 
Grondahl,  247 
Gruber,  211 
Grund,  351 

Haffkine,  222,  320,  321,  351 

Hamburger,  309 

Hamerton,  388 

Hansen,  312 

Harbitz,  247 

Harrison,  361 

Hartmann,  403,  405 

Harvey,  3 

Hauser,  343 

Hellriegel,  14 

Henderson,  297 

Henle,  10,  195 

Herbst,  298 

Herodotus,  7 
^ss,  102,  333 

Hesse,  2 
Heidenhain,  54 

Heim,  164 
Hektoen,  217,  243 
Hill,  35 

Hmdle,  356,  430 
Hippocrates,  8 
Hiss,  51,  258,  337 
Hoffman,  374 
Hoffmann,  E.,  357 
Hogyes,  222 
Holmes,  263 
Homer,  8 
Hornor,  297 
Hubner,  254 
Hutchings,  188 

Irons,  253 
Israel,  246,  247 

Jaeger,  254 

Jeffer,  184,  185 

Jenner,  42 

Jenner,  Edward,  12,  223,  376 


INDEX  OF  NAMES 


441 


Jennings,  434 
Jochmann,  255 
Johns,  423 
Johnson,  179 
Jordan,  180 
Joukoff,  423 

Kanthack,  248 

Keller  man,  182 

Kerr,  J.,  130,  277 

Kilborne,  429 

King,  374 

Kinghorn,  391 

Kircher,  9 

Kirkbride,  39 

Kitasato,  12,  208,  278,  279,  281,  317 

Kitt,  276 

Klebs,  n,  260,  264,  285 

Klegg,  248,  400 

Klimenko,  297 

Knapp,  354 

Kneass,  269 

Koch,  i,  n,  12,  66,  93,  112,  119,  195, 

257,   270,   275,   296,   299,  304, 

305,  330,  345,  347 

Kolle,  221,  222,  223,  302,  321,  351,  368 
Kraus,  209 
Krauss,  39 
Krumwiede,  311,  351 
Kruse,  337 

Laennec,  306 

Lafar,  185 

Lamar,  258 

Landouzy,  306 

Langenbeck  (Von  Langenbeck),  238 

Latzer,  130 

Laveran,  12,  394,  398,  419 

Lazear,  369 

Leeuwenhoek,  3,  5,  9,  15 

Leishman,  42,  335,  356 

Levaditi,  357,  358 

Lewis,  G.  W.,  381 

Lewis,  Paul  A.,  374 

Lewis,  T.  R.,  381 

Liborius,  124 

Lichtheim,  231 

Liebig,  7 

Lindermann,  410 

Lingelsheim    (Von    Lingelsheim),    262, 

265,  280 
Lip  man,  176 
Lister,  n 
Loffler,  2,  52,  285,  287,  308,  329,  339, 

368 

Longley,  181 
Losch,  12,  402 


Lb'wenstein,  305 
Luer,  96 
Luetscher,  327 
Lumbau,  427 

McBryde,  373 

McClintock,  76 

McCrae,  135 

McFadyean,  198 

Mclntosh,  357' 

MacNeal,  43,  102,  117,  193,  311,  378; 

383,  384,  386,  391,  416 
McNeill,  253 
Mackie,  388 
Madsen,  204 
Mafucci,  299 
Major,  260 
Mallory,  93,  254,  297 
Malmsten,  435 
Marchoux,  356 
Marshall,  3  28 
Marzoli,  15 
Massee,  235 
Maurer,  421 
Mayer,  56 
Meltzer,  258 
Mesnil,  394,  398 

Metchnikoff,  212,  218,  225,  227,  330 
Migula;  5,  142,  144,  146 
Milne,  353 
Mique],  75 
Mohler,  341 
MSller,  314 
Montague,  222 
Moore,  179.  182 
Morax,  296 
Moritz,  316 
Moro,  309 
Moses,  7 
Muhlens,  357 
Miiller,  4 
Musgrave,  248,  400 

Nageli,  431 

Needham,  5,  6 

Negri,  370 

Neisser,  215,  250,  265,  312 

Neufeld,  217 

Nicolaier,  278 

Nicolle,  370,  375,  397 

Nocard,  13,  329,  368 

Nocht,  41 

Noguchi,  13,  99,  256,  354,  355,  357,  358, 

359,  366,  367,  375 
Novy,    13,  37,  98,  118,  126,  193,   203, 

326,  328,  354,  378,  383,  384, 

391,  416 


442 


INDEX  OF  NAMES 


Nuttall,  13,  212,  227,  276,  354,  355,356, 
43° 

Obermeier,  n,  13,  353 
Ogston,  260,  264 
Orth,  6 1 

Fakes,  185 
Park,  290,  293,311 
Passini,  145 

Pasteur,  i,  4,  6,  7,  32,  125,  223,  227,  257, 
264,   273,   275,  316,  373,  431, 

432 

Perkins,  C.  F.,  243 
Perkins,  W.  A.,  384 
Petri,  1 08 
Petruschy,  246 
Pettenkofer,  8 

Pfeiffer,  98,  213,  227,  297,  348 
Pirquet  (Von  Pirquet),  219,  309 
Plaut,  240 
Plenciz,  9 
Pollender,  10,  270 
Poor,  370 
Pratt,  351 
Prior,  352 
Prowazek,  378 
Prudden,  188 

Quincke,  255 

Rabinowitsch,  314 
Ramond,  244 
Rattone,  278 
Rayer,  10,  270 
Redi,3 

Reed,  368,  369 
Reichert,  117 
Reimarus,  9 
Remak,  239 
Remlinger,  370 
Rettger,  197 
Ricketts,  244,  375,  376 
Rideal,  77 

Rindfleisch,  n,  260,  264 
Rivolta,  299 
Robin,  10,  238 
Rogers,  121 
Romano wsky,  41 
Rosenau,  283 
Rosenbach,  260,  264 
Rosenow,  367 
Ross,  353,  419,  427 
Rouget,  387 
Rous,  377 

Roux,  119,  288,  293,  368 
Ruediger,  243 


Rufus  of  Ephesus,  319 
RusseU,  334,  335 

Sacharoff,  356 

Sailer,  269 

Salimbini,  356 

Salmon,  13 

Sanarelli,  329 

Sanfelice,  412 

Schafer,  4 

Schaudinn,  357,  358,  392,  403,  405,  408, 

412,  437 
Scheele,  6 
Schenck,  242 
Schereschewsky,  357 
Schonlein,  239 
Schottmiiller,  260 
Schroder,  6 
Schiiffner,  424 
Schulze,  F.,  4,  6 
Schulze,  F.  E.,  400 
Schiitz,  339 
Schwann,  6 
Schwartz,  253 

Schweinitz  (De  Schweinitz),  373 
Sclavo,  274 
Sedgwick,  177,  188 
Seidelin,  370 
Semmelweiss,  263 
Sergent,  Edm.,  396 
Sergent,  Et.,  396 
Shiga,  336 
Sholly,  292 
Siedentopf,  16 
Silberschmidt,  269 
Silbey,  299 
Simon,  364 
Sinton,  389 
Slater,  77 
Smith,  Theobald,  13,  99,  279,  299,  341, 

429 

Sobernheim,  225,  274 
Spall anzani,  4,  5,  6 
Starcovici,  429 
Steinhardt,  370 
Sternberg,  257 
Stevens,  390 
Stewart,  37 
Stribolt,  341 
Strickland,  382 
Strong,  327 
Swellengrebel,  382 

Takaki,  281 
Taylor,  361 
Terre,  299 
Thorn,  139 


INDEX  OF  NAMES 


443 


Thomas,  276 
Thomason,  389 
Tissier,  342 
Todd,  353,  354,  3^8 
Torrey,  378 
Toussaint,  316 
Trudeau,  309 
Tucker,  177 
Tunnicliff,  358,  367 
Tyndall,  6 

Uhlenhuth,  308 

Vallery-Radot,  316,  373 

Van  der  Brock,  7 

Van  Dusch,  6 

Van  Ermengen,  53,  283,  284 

Van  Leeuwenhoek,  3,  5,  9,  15 

Vaughan,  203,  224,  295,  326,  328 

Veillon,  112,  125,  342 

Vianna,  392 

Viereck,  404 

Vignaud,  274 

Villemin,  306 

Vincent,  248,  367 

Von  Behring,  12,  208,  227,  281,  292 

Von  Frisch,  327 

Von  Langenbeck,  238 

Von  Lingelsheim,  262,  265,  280 

Von  Pirquet,  219,  226,  309 

Wassermann,   221,   222,  281,  302,  361, 

368 
Washburn,  87 


Webb,  223 

Wechsberg,  215 

Weeks,  296 

Weichselbaum,  253 

Weigert,  59,  207 

Welch,  51,  267,  276 

Wellman,  312 

Wenyon,  395 

Wertheim,  250 

Wheeler,  188 

Whipple,  181 

Whitmore,  400,  403,  405,  407 

Wilder,  375,  3  76 

Wilfarth,  14 

Williams,  A.  W.,  293,  402,  406 

Williams,  H.  U.,  1 80,  381 

Williamson,  50,  308 

Winslow,  1 88 

Wolf,  247 

Wolff-Eisner,  309 

Wolffhiigel,  185 

Wright,  A.  E.,  217,  228,  266,  335 

Wright,  J.  H.,  125,  248,  254,  396 

Wright,  Jonathan,  327 

Yersin,  288,  317,  321 
Yorke,  391 

Zeiss,  16 
Zettnow,  148 
Ziemann,  392,  415 
Zinsser,  332 
Zsigmondi,  16 


INDEX  OF  SUBJECTS. 


(An  asterisk  (*)  designates  pages  showing  illustrations.) 


Abbe  condenser,  16,  24* 

Aberration,  chromatic,  15 

Abiogenesis,  3,  4 

Abortion,  contagious,  341 

Abrin,  171 

Abscess,  261,  266 

Absorption    of    oxygen    for    anaerobic 

culture,  125 

Accidental  infection,  104 
Acetic  acid,  169 
Achorion  schonleinii,  10,  239,  241* 

cultures  of,  240 
Achromatic  objectives,  16 
Acid,  carbolic,  76 

production  of,  169 
Acid-proof  bacilli,  46,  314 

method  of  staining,  46 
Acids,  germicidal  action  of,  71 
Acne,  342 
Acquired  immunity,  220,  222 

active,  222 

passive,  224 
Actinomyces,  246 

bovis,  246 
Actino  mycosis,  246 
Active  immunity,  222 

duration  of,  222 

methods  of  inducing,  222 
Adaptation  to  environment,  167,  171 

to  parasitism,  202 
Aedes  (Stegomyia)  calopus,  369* 
Aerobes,  166 

sporogenic,  268 
Aerobic  bacteria,  166 
Aerobioscope,  177* 
Agar,  89 

ascitic-fluid,  99 

blood-streaked,  98 

glucose,  91 

glycerin,  91 

sugar,  91 

Age  factor  in  susceptibility,  197 
Agglomerin,  265 
Agglutination,  211,  338 

technic  of,  211 
Agglutinins,  211 
Aggressins,  205 


Agriculture,  14 

relation  of  microbes  to,  14 
Air,  174,  176 

disease-bearing  insects  of,  178 

micro-organisms  of,  176 
Albumen  fixative,  56 
Alcohol,  as  germicide,  78 

production  of,  169 
Alcoholic  fermentation,  169 
Aleppo  boil,  396 
Alexin,  212,  215 
Alimentary  canal,  bacteria  of,  202 

infection,  134 

Alkalies,  germicidal  action  of,  73 
Allergy,  219 
Alopecia  areata,  241 
Alum  in  water  filtration,  181 
Amboceptor,  214 
Ameba  (Amoeba),  155 

cultures  of  saprophytic,  402 

in  tropical  dysentery,  12 
American  filtration,  181 
Ammonia,  170 
Amoeba,  155 

cultures  of  saprophytic,  402 

in  tropical  dysentery,  12 

proteus,  401* 

Amphitrichous  bacteria,  148 
Anaerobes,  166 

sporogenic,  275 
Anaerobic  bacteria,  166 

cultivation  of,  124 

cultures,  124 

Buchner's  method,  125,  126* 

combined  hydrogen  and  pyrogallate 
method,  128*,  129 

deep  stab,  124 

termentation  tube,  125 

in  hydrogen,  126,  127* 

Novy's  method,  126,  127* 

reducing  substances  in,  130 

removal  of  oxygen,  125 

under  paraffin,  130 

Veillon  tube,  124 
Anaphylaxis,  226 
Aniline  dyes,  40 

disinfectant  action  of,  78 


445 


446 


INDEX  OF  SUBJECTS 


Aniline-water  staining  solutions,  40 
Animal  experimentation,  131 

value  of,  131 
Animals,  care  of,  131 

experimentation  with,  131 

holding  of,  132 

inoculation,  133 

observation  of  infected,  136 
Anopheles  mosquitoes,  427,  428* 
Anthrax,  12,  270,  272 

bacillus,  270 

colony,  271 

immunity,  273 

infection,  273 

intestinal,  273 

pulmonary,  273 

pustule,  273 

serum,  274 

vaccine,  273 
Antiaggressins,  218 
Antibacterial  serum,  212,  213 
Antibodies,  206,  208,  218 

distribution  of,  218 

source  of,  218 

Anticomplementary  reaction,  363 
Antiformin  method,  50 
Antigen,  217,  228 
Antimeningococcus  serum,  255 
Antipneumonococcus  serum,  260 
Antisepsis,  62,  79 
Antiseptic  surgery,  n 
Antiseptics,  79 

testing  of,  80 

Antistreptococcus  serum,  264 
Antitoxic  serum,  208 
Antitoxin,  diphtheria,  12,  208,  292,  293 

concentration  of,  293 

curative  value  of,  295 

preparation  of,  293 

prophylactic  value,  295 

standardization  of,  294 
Antitoxin,  tetanus,  12,  208,  281 
Antityphoid  vaccination,  335 
Apochromatic  objectives,  16 
Arnold  steam  sterilizer,  67*,  68* 
Arthritis,  streptococcus,  264 
Artificial  culture,  163 
Ascitic  fluid,  sterile,  97 

agar,  99 

with  sterile  tissue,  99 
Ascomycetes,  137 
Asiatic  cholera,  345,  349 

cairiers  of,  351 

diagnosis,  350 

epidemics  of,  348 

history  of,  348 

prophylaxis,  351 


Asiatic  quarantine  in,  351 

spirillum  of,  345 

transmission  of,  349 

vaccine  for,  351 
Aspergillosis,  233 
Aspergillus  fumigatus,  233 

glaucus,  138*,  233* 
Atmosphere,  bacteria  of,  176 

hydrogen,  for  anaerobes,  126 
Atrichous  bacteria,  148 
Attenuation,  202 
Autoclave,  68,  69* 

sterilization,  68 
Autopsies,  103 
Avenues  of  infection,  197 
Avian  tuberculosis,  299,  311 
Avoidance  of  contamination,  104 
Azotobacter,  175 
Azure,  methylene,  42,  43 

Babesia,  155,  158*,  429* 

bigemina,  429* 
immunity  to,  430 
transmission  of,  430 

canis,  430 

muris,  158* 
Bacilli,  145* 

acid-proof,  46,  299 

capsulated,  51,  148,  326,  338 

chromogenic,  343 

colon- typhoid,  324 

pigment-producing,  343 
Bacillus,  5,  142,  144 

abortus,  341 

acne,  342 

aerogenes,  326 

aerogenes    capsulatus  (B.    welchii), 
276 

alkaligenes,  329 

anthracis,  10,  12,  270* 

anthracis  symptomatici  (feseri),  276 

ayisepticus,  316 

bifidus,  342 

Bordet-Gengou  (B.  pertussis),  296 

botulinus,  283 

bulgaricus,  342 

butter,  314 

capsulatus,  328 

chancri,  298 

chauvei  (B.  feseri),  276 

cholerse-suis  (B.  suipestifer),  329 
Bacillus  coli,  324* 

cultures,  325 

detection  of,  326 

in  water  supplies,  186 

pathogenic  properties,  326 

poisons  of,  326 


INDEX  OF  SUBJECTS 


447 


Bacillus,  comma  (Sp.  cholerae),  345 

cyanogenus,  343 
Bacillus,  diphtherias,  285 

animal  inoculation,   285,   288,    291 

bacilli  resembling,    289,    292,    295, 
296 

cultural  characters,  287 

granular  types,  285 

in  human  body,  290 

Loffler's    serum    for    culture,    287, 
290*,  291 

mode  of  infection,  292 

morphology,  285 

resistance,  288 

solid  types,  285 

staining  of,  286,  291 

toxin  of,  288 

types,  286*,  287* 

virulence,  291 
Bacillus,  Ducrey's,  298 

dysenteric,  336 

edematis,  275 

enteritidis,  328 

fecalis  alkaligenes,  329 

feseri,  276 

fluorescens,  343 

fusiformis    (Spirochaeta    vincenti), 

367 

Gartner's  (B.  enteritidis),  328 
gas  (B.  welchii),  276 
grass  (B.  molleri),  314 
hay  (B.  subtilis),  269* 
hoffmanni,  296 
icteroides,  329 
influenzas,  297 

Klebs-LOfikr  (B.  diphtherias),   285 
Koch-Weeks,  296,  297* 
lactici-acidi,  190 

lactis  aerogenes  (B.  aerogenes),  326 
leprae,  3 1 2 
mallei,  339* 
melitensis,  321 
mesentericus,  268 
molleri,  314 
Morax-Axenfeld,  296* 
mucosus,  328 
murisepticus,  317 
mycoides,  268 
ozenae,  328 
paracolon,  330 
paradysentery,  337 
paratyphoid,  330 
perfringens  (B.  welchii),  276 
pertussis,  296 
Bacillus  pestis,  317* 
cultures,  318 
immunity,  320 


Bacillus  pestis  in  animals,  319 
morphology,  317* 
toxins,  318 

Bacillus  plurisepticus,  316 
pneumoniae,  327 
potato  (B.  vulgatus),  268 
prodigiosus,  344 
proteus,  343 
pseudo-diphtheria   (B.   hoffmanni), 

296 

psittacosis,  329 
pyocyaneus,  343 
radicicola,  14 
rhinoscleromatis,  327 
rhusiopathiae  suis,  317 
salmonii,  329 

Shiga's  (B.  dysenteriae),  336 
subtilis,  269* 
suipestifer,  329 
tetani,  278 
Bacillus  tuberculosis,  299 

amphibian,  312 

avian,  311 

bovine,  310 

branching  of,  302* 

chemical  composition  of,  302 

cultures  of,  301 

fish  type,  312 

human  type,  300* 

morphology,  300 

poisons  of,  303 

resistance  of,  304 

varieties  of,  299 
Bacillus  typhi  murium,  329 
Bacillus  typhosus,  330 
agglutination  of,  334 
distribution  of,  330,  332,  333 
flies  as  carriers  of,  335 
human  carriers  of,  333,  335 
in  blood,  333 
in  feces,  333,  334 
in  food,  335 
in  milk,  335 
in  soil,  335 
in  sputum,  333 
in  urine,  333 
in  water,  187,  335 
isolation  of,  354 

pathogenic  properties  of,  330,  332 
poisons  of,  332 
resistance  of,  332 
vaccines,  335 
Bacillus  violaceus,  343 
vulgaris,  343 
vulgatus,  268 
welchii,  276 
xerosis,  295 


448 


INDEX  OF  SUBJECTS 


Bacteremia,  200 

streptococcus,  264 
Bacteria,  141 

acid-proof,  46,  299 

adaptability,  167 

aerobic,  166 

anaerobic,  166,  275 

classification,  137,  141,  160 

colonies,  109*,  172 

cylindrical,  144 

dimensions,  141 

discovery,  3 

distribution,  174 

fluctuation,  167 

food,  189 

in  air,  174,  176 

in  agriculture,  175 

in  food,  189 

in  ice,  178 

in  milk,  189 

in  soil,  175 

in  water,  178 

soil,  175 

spherical,  142 

spiral,  146 

structure  of,  147 

variation,  167 

with  spores,  149* 
Bacteriaceae,  144,  268 
Bacterial  poisons,  171,  203 

vaccines,  12,  223 
Bactericidal  substances,  212,  227 
Bacteriology,  i 

biological  relations,  3 

history,  i 

hygienic,  7 

nomenclature  of,  160 

scope,  2 

Bacteriolysins,  213 
Bacteriolysis,  213 
Bacterium,  5,  144 
Balantidium  coli,  435 

parasitic  relations,  435,  436* 

pathogenesis,  436 
Balantidium  minutum,  437 
Basic  dyes,  40 
Basidiomycetes,  139 
Basophile  granules,  60 
Bed-bugs  (Cimex),  395 
Beef-tea  (broth),  84 
Berkefeld  filter,  63 
Bichloride  of  mercury,  74 
Biological  relationships  of  bacteriology, 

3 

Birds,  malaria  of,  412,  414,  415,  417 
trypanosomes  of,  390* 
tuberculosis  of,  299,  311 


Black  death  (plague),  317 
Black-leg,  276 

Blastomyces  dermatidis,  245 
Blastomycetes,  140*,  244 
Blastomycetic  dermatitis,  245 
Blastomycosis,  244 
Bleaching  powder,  74 
Blepharoplast,  378 
Blood-agar,  Novy's,  98 

Pfeiffer's,  98 
Blood,  92 

bacteria  in,  200 

citrated,  96 

culture,  101 

defibrinated,  96 

films  for  microscopic  examination, 

54 

protozoa  in,  200 

sterile,  collection  of,  95 
Blood  serum,  92 

as  culture  medium,  92 
Loeffler's,  84 
Blue  milk,  343 

PUS,  343 

Bodo  lacertae,  398* 
Boil,  Delhi,  396 
Boils,  266 

Boophilus  bovis,  158,  429 
Bordet-Gengou  bacillus  (B.  pertussis), 

296 

Boric  acid,  79 
Botrytis  bassiana,  10,  235 
Botulin,  284 
Botulism,  284 

antitoxin  for,  284 
Bouillon  (broth),  84 
Bovine  pleuro-pneumonia,  368 

tuberculosis,  310 
Branching  bacilli,  302* 
Bread-paste,  94 
Bromine,  74 
Broth,  nutrient,  84 

containing  sterile  tissue,  99 

sugar,  90 

sugar-free,  90 
Brownian  movement,  34 
Bubonic  plague,  317,  319 

diagnosis,  318 

fleas  as  carriers  of,  319 

history  of,  319 

immunity  to,  320 

prophylaxis  of,  320,  321 

rodents  as  reservoirs  of,  314,  320 

serum,  321 

vaccines,  320 

Buchner  method  for  anaerobic  culture 
125 


INDEX  OF  SUBJECTS 


449 


Burner,  Bunsen,  105 

Koch's  automatic  safety,  119*,  120 
Butter  bacillus,  314 
Butyric  acid  test,  256 

Calcium  oxide  (lime),  73 

Calmette's  test  (tuberculin),  309 

Capsules,  147,  148* 

Carbol-fuchsin,  41 

Carbolic  acid,  76 

Carbuncles,  266 

Carmine,  61 

Carriers  of  infection,  201 

Caseation,  306 

Cattle  plague,  370 

tick,  158,  429 
Cedar-wood  oil,  30 
Cell,  chemical  constitution  of,  207 
Cell-membrane  of  bacteria,  147 
Celloidin,  55 

Cell-receptors,  209*,  210*,  214* 
Cerebro-spinal  fluid,  255 

collection  of,  101,  255 

examination  of,  256 

in  meningitis,  255 

test  for  globulins  in,  256 
Chancroid,  258 
Charbon  (Anthrax),  270,  272 
Cheese,  191,  237 
Chemical  agents  as  germicides,  71 

disinfection,  72,  77 

effects,  168 

products,  1 68 
Chicken  cholera,  316 

sarcoma,  377 
Chlorine,  73 

Chloroform,  preservative  action  of,  79 
Cholera,  Asiatic,  345 

carriers,  351 

diagnosis  of,  350 

prophylaxis,  351 

transmission  of,  349 
Cholera,  fowl,  316 
Cholera,  hog,  373 
Chromatin,  148 
Chromogenic  bacteria,  343 
Ciliates,  159,  433* 
Ciliophora,  151,  159,  433* 
Cladothrix,  246,  249 
Classification,  4,  137 

of  molds,  137 

of  protozoa,  150 

of  yeasts,  140 

outline  of  micro-organisms,  160 
Claviceps  purpurea,  234 
Cleaning  fluid,  37 

Clearing  microscopic  preparations,  28 
29 


Clostridium,  146* 

Coccaceae,  142*,  250 

Cocci,  142*,  250 

Cocci dioidal  granuloma,  245 

Coccidiosis,  411 

Coccidium  (Eimeria),  155,  156* 

cuniculi  (Eimeria  steidae),  410 
Coccus,  142 

Cold,  effect  on  bacteria,  64 
Collection  of  material,  lo'o 

of  sterile  ascitic  fluid,  97 

of  sterile  blood,  95 

of  sterile  tissue,  98 
Collodion  capsules,  134,  135* 

embedding,  55 
Colon  bacillus,  324* 

cultures  of,  325 

detection  of,  326 

in  water-supplies,  186 

pathogenic  properties  of,  326 

poisons  of,  326 

Colonies  of  bacteria,  ic6,  109*,  113 
Comma  bacillus,  345 
Commensal.  194 
Complement,  215,  216 

deviation  of,  215 

detection  of,  217 

fixation  of,  216,  361 
Complement-fixation  test,  361 

antigen,  363 

blood  cells  for,  561 

complement  for,  361 

hemolytic  amboceptor  for,  362 

patient's  serum  for,  363 

signification  of,  366 

technic  of,  364 
Condenser,  sub-stage  (Abbe).  24* 

dark  field,  25* 
Conjunctivitis,  296,  297 
Conorhinus  megistus,  395* 

as  vector  of  cereotrypanosis,  394 
Consumption  (tuberculosis),  305 
Contagion,  200 

early  ideas  of,  7,  8,  9 
Contagious  abortion,  341 

disease,  200 

Contamination,  avoidance  of,  104 
Coreotrypanosis,  392 

transmission  of,  394 
Cornet  forceps,  38* 
Corrosive  sublimate,  74 
Cotton  plugs,  84 
Cover-glass  forceps,  37,  38* 

preparations,  36 
Cover-glasses,  36 

cleaning  of,  36 
Cow-pox,  12,  376 


45° 


INDEX  OF  SUBJECTS 


Creolin,  77 

Cresol,  77 

Croupous  pneumonia,  259 

Culex  mosquitoes,  427,  428* 

Cultivation,  104 

of  anaerobes,  124 

of  bacteria,  104 

of  protozoa,  13 

of  spirochetes,  13 
Culture  media,  83 

agar,  89 

bread-paste,  94 

broth,  84 

blood-agar,  98 

blood-serum,  92 

choice  of,  115 

containing  uncooked  protein,  95 

dextrose,  90 

dextrose-free,  90 

Dorset's  egg,  94 

Dunham's  solution,  92 

filling  into  tubes,  89* 

gelatin,  88 

lactose,  90 

litmus,  90 

Loffler's  blood  serum,  94 

method  of  inoculating,  107* 

milk,  92 

nitrate-broth,  92 

peptone  solution,  92 

potato,  91 

preparation  of,  84 

special,  91 
Cultures,  plate,  12,  106 

pure,  113 

roll-tube,  no*,  in* 

sealing  of,  120 

smear,  113,  114* 

stab,  114*,  124 

stock, 114 

streak,  113,  114* 

tube,  112,  114*,  124 
Cutaneous  tuberculin  test,  309 
Cutting  sections,  56 
Cyclospora  caryolytica,  408* 

life  cycle  of,  408 

parasitic  relations,  408 

pathogenesis,  410 
Cystitis,  327 
Cytase,  215 
Cytolysins,  213 
Cytolysis,  213 

Dark-field  microscopy,  16,  35 
Decomposition,  5,  6,  7,  170 
Deep  stab-cultures,  124 
Defects  of  lenses,  20 


Defensive  mechanisms  of  microbes,  204 

Delafield's  hematoxylin,  61 

Delhi  boil,  396 

Deneke's  spirillum,  352 

Dengue  fever,  374 

Denitrification,  175 

Descriptive  chart,  Soc.  Am.  Bact.,  174 

Desiccation,  63,  164 

Development  of  bacteriology,  i 

Deviation  of  complement,  215 

Dextrose,  fermentation  of,  169 

media,  90 

Dextrose- free  media,  90 
Diffraction,  20 

Dilution  cultures,  105,  106,  no,  112 
Dimensions  of  bacteria,  141 
Diphtheria,  289 

antitoxin,  273,  293 

bacillus  (B.  diphtheriae),  285 

carriers,  292 

diagnosis,  290 

immunity  to,  292 

mixed  infection,  290 

prophylaxis  of,  295 

streptococcus  in,  290 

toxin,  288 

transmission  of,  292 
Diplobacillus,  145 

of  Morax-Axenfeld,  296* 
Diplococcus,  142* 

catarrhalis,  257 

gonorrheae,  250 

meningitidis,  253 

pneumoniae,  257 
Disease,  causation  of,  195 

contagious,  8 

infectious,  196 

miasmatic,  8 

phenomena  of,  195 

theories  of,  9,  206 
Disinfectants,  71 

testing  of,  80 
Disinfection,  62 

of  feces,  73 

of  rooms,  72,  77 
Distribution  of  micro-organisms,  174 

in  animal  body,  175 

in  atmosphere,  174,  176 

in  foods,  189,  193 

in  milk,  189 

in  plant  tissues,  175 

in  soil,  175 

in  water,  174,  178 
Diversion  of  completement,  215 
Doflein's  classification  of  protozoa,  150 
Dorset's  egg-medium,  94 
Dourine,  387 


INDEX  OF  SUBJECTS 


451 


Drinking  water,  178 

Drying  (desiccation),  63 

Ducrey's  bacillus,  298 

Dum-dum  fever  (kala-azar),  394,  396 

Dunham's  peptone  solution,  92 

Dust,  176 

Dyes,  40 

acid,  40 

aniline,  40,  78 

basic,  40 
Dysentery,  ameba  of,  404 

amebic,  406 

entamebic,  406 

bacillary,  336 

bacillus,  336 

diagnosis,  336 

serum  therapy,  337 

Eberth's  bacillus,  330 

Edema,  malignant,  276 

Egg,  fresh,  as  culture  medium,  94 

Dorset's  egg  medium,  94 
Ehrlich's  conception  of  chemical  struc- 
ture of  cell,  207 

side  chain  theory,  208,  227 

theory  of  immunity,  227 
Eimeria,  155,  156* 

schubergi,  156*,  412 
Eimeria  steidae,  410* 

life  cycle  in  rabbit,  410 

pathogenesis,  411 

Electricity,  germicidal  action  of,  71 
Electric  thermostat,  122 
Emmerich's  bacillus  (B.  coli),  324* 
Emphysematous  gangrene,  277 
Encapsulation,  207 
Endocarditis,  260 

lenta,  260 

staphylococcus,  266 

subacute,  260 
Endo's  medium,  354 
Endospores,  146,  149,  155 
Endotoxins,  204 
Entamceba,  154*,  155 
Entamceba  coli,  154*,  402* 

morphology,  403 

occurrence,  402 

parasitic  relation  of,  403 
Entamceba  histolytica,  405 
Entamceba  tetragena,  404* 

cyst  of,  405* 

morphology,  404* 

questionable  cultures  of,  406 

relation  to  dysentery,  405,  406 

transmission  of,  405 
Entamcebic  dysentery,  406 
Enteric  fever  (typhoid  fever),  333 


Environment,  164 

mutual  relation  of  microbe  and,  171 
Enzymes,  169 

diastatic,  170 

glycolytic,  ^170 

proteolytic,  170 

steatolytic,  171 
Eosin,  40 

Epibemic  meningitis,  253 
Epithelioid  cells,  306 
Erysipelas,  263 

immunity  to,  264 

streptococci  in,  260 
Escherich's  bacillus  (B.  coli),  324 
Esmarch  roll-tubes,  no*,  in* 
Estivo-autumnal  malaria,  419,  426 
Extracellular  toxins,  203 
Exudates,  102 
Eye-piece,  23,  29,  30 

narrowing  of  the  beam  by,  23 

Facultative  serobe,  166 

anaerobe,  166 

Fatigue  predisposing  to  infection,  197 
Fats,  fermentation  of,  171 
Favus,  10,  239*,  240* 
Feces,  collection  for  examination,  102* 

disinfection  of,  73 

typhoid  bacilli  in,  354 
Fermentation,  5,  6,  7,  169 

acetic,  169 

alcoholic,  169 

of  milk,  191 

tube,  125,  186 
Film  preparations,  36 

of  blood,  54 
Filters,  Berkefeld,  63 

Pasteur-Chamberland,  63 

sand,  1 80 

Filterable  micro-organisms,  13,  150,  368 
Filterable,  virus,  13,  150,  368 

of  bovine  pleuro-pneumonia,  368 

of  cattle  plague,  370 

of  chicken  sarcoma,  377 

of  dengue  fever,  374 

of  foot-and-mouth  disease,  368 

of  hog  cholera,  373 

of  measles,  375 

of  phlebotomus  fever,  374 

of  pleuro-pneumonia,  368 

of  poliomyelitis,  374 

of  rabies,  3  70 

of  rinderpest,  370 

of  small-pox,  376 

of  typhus  fever,  375 

of  yellow  fever,  368 
Filtration  of  bacterial  cultures,  63 


452 


INDEX  OF  SUBJECTS 


Filtration  of  media,  85,  88,  90,  97 

of  water,  180 

sterilization  by,  63,  97 
Finkler  and  Prior,  spirillum  of,  352 
Fishing  colonies,  113 
Fixation  of  complement,  216 

in  glanders,  341 

in  gonorrhea,  253 

in  syphilis,  361 
Fixation  of  protozoa,  54 
Fixative,  albumen,  56 
Flagella,  bacterial,  148* 

staining  of,  52 

Flagella  of  protozoa,  151,  153* 
Flagellates,  151 
Fleas,  319,  382,  396 
Flies,  154,  335,  387,  388 
Fluctuating  characters,  4 
Focusing,  30 
Fomites,  200 
Food,  bacteria  in,  189,  193 

of  micro-organisms,  164 

poisoning,  195,  284,  328 

preservation,  6,  62,  79,  193 
Foot-and-mouth  disease,  368 

filterable  virus  of,  368 
Forceps,  cover-glass,  37,  38* 
Formaldehyde,  77 
Formalin,  77 
Fowl-cholera,  316 

tuberculosis,  311 
Fractional  sterilization,  70 
Fraenkel's  diplococcus,  257 

cultures  of,  258 
Freezing,  64,  166 
Friedlander's  bacillus,  257,  327 
Fuchsin,  40 

carbol-,  41 
Furuncle,  266 
Fusiform  bacillus,  367 

Gametes,  156* 
Gametocytes,  155,  156* 
Gangrene,  emphysematous,  277 
Gartner's  bacillus  (B.  enteritidis),  328 
Gas  bacillus  (B.  welchii),  276 
Gas-burner,  Koch's  safety,  119*,  120 
Gas-formation,  169 
Gas-regulator,  116,  117*,  118*,  119* 
Gastric  juice,  gerroicidal  action  of;  71 
Gelatin  cultures,  106,  113,  114* 

nutrient,  88 

plates,  1 06 
Gentian  violet,  40 
Germ  carriers,  201 
Germicidal  serum,  212 
Germicides,  71,  78 


Giemsa's  stain,  42 
Glanders,  340 

bacillus,  339 

diagnosis,  340 
Glassware,  83 
Glossina  morsitans,  385*,  386 

palpalis,  388,  389* 
Glucose  media,  90 
Glycerin  media,  90 
Gonococcus,  250 

cultures  of,  251 

in  pus,  251* 
Gonorrhea,  252 

diagnosis  of,  253 

prophylaxis  of,  253 
Gonorrhea!  arthritis,  252 

ophthalmia,  252 

vulvo-vaginitis,  252 
Gram-negative  bacteria,  45 
Gram-positive  bacteria,  46 
Gram's  stain,  44 

Gram-Weigert  method  of  staining,  59 
Granuloma,  coccidioidal,  245 
Grass  bacillus  (B.  molleri),  314 
Green  pus,  343 

Gregarina  blattarum,  430,  431* 
Gruber-Widal  reaction,  211,  338 

Hsemoproteus  columbae,  412 

distribution,  412 

life  cycle  of,  42,  413* 

transmission  of,  412 
Haemoproteus  danilewskyi,  414* 

ziemanni,  415*,  416* 
Haffkine's  prophylactic  inoculation,  320 

for  cholera,  351 

for  plague,  3  20 
Halteridium,  414* 
Hanging-block,  35 
Hanging-drop,  33,  34* 
Haptophore,  204,  211 
Hardening  of  tissues,  55 
Hay  bacillus  (B.  subtilis),  269* 
Healthy  carriers  of  infection,  201 
Heat  in  food  preservation,  6 

production  of,  168 

separation  of  bacterial  species  by, 
'  .278,^279 

sterilization  by,  64 
Hematoxylin,  Delafield's,  61 

Heidenhain's  iron,  54 
Hematozoa,  379,  412 
Hemolysins,  216 
Hemolysis,  216 
Hemolytic  amboceptor,  216 

serum,  216 

titration  of,  216 


INDEX  OF  SUBJECTS 


453 


Hemorrhagic  septicemia,  316 
Herpetomonas  muscae,  378 

culicis,  378 
Heterogenesis,  4 

High-temperature  incubator,  115,  116* 
Higher  bacteria  (trichobacteria),  141 
Hiss  capsule  stain,  51 

serum- water  medium,  377 
History  of  bacteriology,  i 
Hoffmann's  bacillus  (B.  hoffmanni),  296 
Hog  cholera,  373 

bacillus  of  (B.  suipestif er) ,  329 

immunity,  374 

serum,  374 

spirochete,  374 

virus  of,  373 

Hogyes  treatment  of  rabies,  222 
Honing  of  knives,  57 
Hot-air  sterilization,  64 

sterilizer,  65* 
House  disinfection,  72,  77 
Hunger  predisposing  to  infection,  197 
Hydrochloric  acid  as  germicide,  7 1 
Hydrogen  atmosphere  for  anaerobes,  126 

peroxide,  74 
Hydrophobia  (rabies),  370,  372 

diagnosis,  372 

Negri  bodies  in,  370,  371 

Pasteur  treatment,  373 

treatment  of  wound,  373 
Hyperplasia,  206 
Hypersusceptibility,  226 
Hypha,  137 
Hyphomycetes,  137 
Hypochlorite,  73,  182 
Hypodermic  inoculation,  133 

Ice,  1 88 

bacteriological  examination  of,  188 
Illumination  by  broad  beam,  24* 

by  hollow  cone,  24*,  25* 

central,  24* 

dark  field,  24*,  25* 
Image  formation,  17*,  18* 
Imbedding,  55 
Immune  body,  2I41 
Immunity,  12,  220 

acquired,  220,  222 

active,  222 

antiaggressive,  224 

antitoxic,  224 

bacteriolytic,  213 

combined  passive  and  active,  225 

duration  of,  222 

Ehrlich's  theory  of,  227 

following  vaccination,  223 

individual,  221 


Immunity,  mechanisms  of,  225 

natural,  220 

of  species,  220 

passive,  224 

racial,  221 

theories  of,  227 

unit,  281,  294 
Impression  preparation,  37 
Inactivated  serum,  213,  363 
Incubator,  115,  116* 

low  temperature,  121 

rooms,  1 20 
Infection,  196 

avenues,  197 

general,  199 

healthy  carriers  of,  201 

local,  199 

possibility  of,  196 

secondary,  199 

transmission  of,  197,  200 
Infectious  disease,  196 

facts  and  theories  of,  206 

phenomena  of,  206 
Influenza,  298 
Inoculation,  animal,  133 

into  the  circulating  blood,  133 

into  the  cranial  cavity,  133 

intracardiac,  133 

intraperitoneal,  133 

subcutaneous,  133 

Inorganic  salts  as  microbic  food,  165 
Insects,  13 

destruction  of,  72 
Instruments,  sterilization  of,  66 
Intermediary  body,  214 
Intermittent  sterilization,  70 
Intestinal  amebae  (entamcebae),  402 

anthrax,  273 

juice,  collection  of,  102 
Intestine,  infection  through,  199 
Intrauterine  infection,  198 
Intravenous  inoculation,  133 
Invisible  microbes,  9,  26 
Iodide  of  mercury,  75 
Iodine,  74 

antiseptic  value,  79 
lodoform,  74 
Iris  diaphragm,  24 
Iron  hematoxylin,  54 
Isolation  of  bacteria,  105,  112 

plate  method,  105 

streak  method,  112 

Veillon  method,  112 
Itch  (scabies),  10 

Jaw,  lumpy  (actinomycosis),  246 
Jeffer's  plate,  184 


454 


INDEX  OF  SUBJECTS 


Jennerian  vaccination,  223,  376 
Jenner's  stain,  42 

Kala-azar,  394,  396 

parasite  of,  394 

transmission  of,  395 
Kefir,  192 

Kirkbride  forceps,  39* 
Klebs-Loffler  bacillus,  285 
Koch-Eberth  bacillus,  330 
Koch's  safety  burner,  119* 

plate  cultures,  106 

postulates,  195 

steam  sterilizer,  66 
Koch- Weeks  bacillus,  296,  297* 
Koumiss,  192 

Lactic  acid,  169 
Lamblia,  153 

intestinalis,  153*,  399*,  400 
Leishman-Donovan    bodies    (L.    dono- 

vani)',  152*,  394 
Leishmania  donovani,  152*,  153*,  394 

cultures,  394 

occurrence,  394 

transmission,  395 
Leishmania  infantum,  397 
Leishmania  tropica,  396* 

cultures  of,  397* 

immunity  to,  397 
Leishman's  stain,  42 
Leprosy,  313 
Leptomonas  culicis.  378 
Leptothrix,  246,  249 

buccalis.  249 
Leukoddin,  265 
Levaditi's  silver  stain,  358 
Light,  effect  on  bacteria,  63 
Lime,  73 

Lithium  carmine,  61 
Litmus,  85,  87 
Lockjaw  (tetanus),  278,  280 
Locomotion,  34 
Loffler's  bacillus  (B.  diphtheria?) ,  285 

blood  serum,  94 

flagella  stain,  52 

methylene  blue,  41 
Lophotrichous  bacteria,  148 
Lower  bacteria,  142 
Luetin,  359 

test,  366 
Lumpy  jaw,  246 
Lungs,  infection  of,  198 

inflammation  of  (pneumonia),  259 
Lysins,  213 
Lysol,  77 
Lyssa  (rabies),  370,  372 


Lyssa  (rabies),  diagnosis  of,  372 
Hogyes  treatment  of,  222 
Pasteur  treatment  of,  373 

Macrogametes,  156* 
Macrogametocytes,  156* 
Madura  foot,  248 
Madurella  mycetori,  249 
Magnification,  16*,  17*,  18*,  19,  23 
Malachite  green,  78 
Malaria,  425 

ayian,  412,  414,  415,  417 

diagnosis  of,  427 

estivo-autumnal,  419,  426 

mosquitoes  in,  427,  428 

prophylaxis,  427 

quartan,  425,  426*,  427 

tertian,  424*,  426 

transmission  of,  427 
Malarial  parasites   of   birds,  412,  414, 

415,  4i7 

of  man,  419,  424*,  425,  426* 

of  monkeys,  429 

transmission  of,  472 
Mai  de  Caderas,  388 
Malignant  edema,  276 

pustule,  273 
Mallein,  340 
Malta  fever,  321,  322 

diagnosis  of,  322,  323 
Mammalian  tuberculosis,  299 
Marmorek's    serum    (antistreptococcus 

serum),  264 

Mastigamceba  aspera,  400 
Mastigphora,  151,  3  78 
Mayer's  glycerin-albumen,  56 
Measles,  375 
Mechanical  filtration,  181 

sterilization,  62 
Media,  culture,  83 
Mediterranean  fever  (Malta  fever),  321, 

322 

Membranous  croup  (diphtheria),  289 
Meningitis,  253 

diagnosis,  255,  257 

serum,  255 

serum  treatment,  255 
Meningococcus,  253,  256* 

cultures  of,  254 
Mercuric  chloride,  74 

iodide,  75 
Metchnikoff's  phagocytic  theory,  225, 

227 

Methyl  violet,  78 
Methylene  azure,  42,  43 
Methylene  blue,  41 

germicidal  power  of,  78 


INDEX  OF  SUBJECTS 


455 


Methylene  violet,  43 
Miasm,  200 
Microbe,  3 

relation  of,  to  environment    171 
Microbiology,  3 
Micrococcus,  5,  142,  143 

agilis,  267 

catarrhalis,  257 

gonorrhea?,  250 

melitensis,  321 

meningitidis,  253 

tetragenus,  267 
Microgamete,  156* 
Microgametocyte,  156* 
Micro  millimeter,  31 
Micron,  31 
Micronucleus,  433 
Micro-organisms,  3 

distribution  of,  174 

in  air,  176 

in  food,  193 

in  ice,  188 

in  milk,  189 

in  soil,  175 

in  water,  178 
Microscope,  15,  21*,  29* 

development  of,  15 

principle  of,  16,  22* 

tandem,  16 

use  of,  31 
Microscopic  definition,  24 

measurements,  31 

resolution  in  depth,  24 
Microspira,  146 

comma,  345 
Microsporon  audouini,  241 

furfur,  242 

septicum,  n,  260 
Microtome,  57* 

Migula's  classification  of  bacteria,  142 
Miliary  tuberculosis,  307 
Milk,  189 

acid,  beverages,  343 

as  culture  medium,  92 

bacteria  of,  189 

blue,  344 

collection  of  samples  of,  100 

composition  of,  189 

for  infant  feeding,  192 

pasteurization  of,  192 

micro-organisms  of,  190 
Milzbrand  (anthrax),  270,  272 
Mixed  infection,  199 
Modes  of  entry  of  infection,  197 
Moisture      requirement     of     bacteria, 

164 
Molds,  137,  231 


M  oiler's  grass  bacillus  (Bacillus  molleri), 

3H 

spore  stain,  51 
Monilia  Candida,  238* 
Monotrichous  bacteria,  148 
Morax-Axenfeld  bacillus,  296* 
Morphology,  137 

relation  of,  to  environment,  171 

relation  of,  to  physiology,  162 
Mosquitoes  in  malaria,  427,  428* 
Motility,  34 
Movement,  34 

Brownian,  34 

real,  34 
Mucor,  231,  232*,  233 

corymbifer,  231,  232* 

mucedo,  138*,  231,  232* 
Muscardine,  10,  236 
Musgrave    and    Clegg's    medium    for 

ameba,  402 
Mycelium,  137 
Mycetoma,  248 

Nagana,  384,  386 

diagnosis  of,  387 

immunity  to,  387 

occurrence  of,  386 

transmission  of,  386 

trypanosome  of,  384* 
Natural  immunity,  220 

individual,  221 

mechanisms  of,  225 

of  species,  220 

racial,  221 

Negri  bodies,  370,  371 
Neisser's  gonococcus,  250 
Neisser-Wechsberg  phenomenon,  215 
Neosporidia,  158,  431 
Neutralization  of  culture  media,  85,  87 
Nitrate  of  silver,  76 

Nitrates,  production  of,  by  bacteria,  175 
Nitrification,  175 
Nitrifying  bacteria,  175 
Nitrites,  formation  of,  175 
Nitrogen  fixation,  175 
Nitrosomonas,  164 
Nocht-Romanowsky  stain,  41 
Nodule  bacteria  (root   tubercles),    175, 

194 

Nomenclature,  160 
Normal  solution,  85 
Nosema,  155,  158 

bombycis,  n,  159*,  431,  432* 
Novy's  anaerobic  method,  126,  127* 

blood-agar,  98 

cover-glass  forceps,  38* 
Nuclear  stains,  61 


456 


INDEX  OF  SUBJECTS 


Nucleus  of  bacteria,  148 

of  protozoa,  151 
Number  of  bacteria  in  milk,  189 

in  water,  183 

required  to  infect,  197 
Numerical  aperture,  23 
Nutrient  agar,  89 

Obermeier's  spirillum,  353 
Objectives,  achromatic,  16,  20*,  29,  30 

apochromatic,  16,  20 

defects  of,  20 

immersion,  30 

Ocular  tuberculin  reaction,  309 
Oculars,  23,  29,  30 
Oidiomycosis,  245 
Oidium  albicans,  10,  238* 

lactis,  138*,  236* 
Oil,  aniline,  in  stains,  40 
Ookinete,  157 

Opalina  ranarum,  434,  435* 
Opsonins,  217 
Organic  poisons  as  germicides,  76 

food  requirements,  164 
Oriental  sore  (Delhi  boil),  396 
Osteomyelitis,  266 
Outline  classification,  i6q 
Ovum,  infection  of,  197 
Oxidizing  agents  as  germicides,  73 
Oxygen,  166 

requirement,  166 

removal  of,  1 25 
Oysters  as  source  of  typhoid,  335 

Panophthalmitis,  269 
Paracolon  bacilli,  330 
Paraffin,  55 

imbedding,  55 
Paralysis,  infantile,  374 
Paramaecium  aurelia,  434 

bursaria,  434 

caudatum,  433 

conjugation,  433,  434 

division,  434 

form  and  structure,  433 
Paramascium  putrinum,  434* 
Paraplasma  flavigenum,  370 
Parasite,  165 

obligate,  165 
Parasitism,  194 
Paratyphoid  bacilli,  330 
Parenteral  digestion,  207 
Passive  immunity,  224 
Pasteur  pipettes,  33 

treatment  for  rabies,  373 
Pasteur-Chamberland  filter,  63 
Pasteurization,  66 


Pathogenesis,  194,  195 
Pathogenic  bacteria,  195 

organisms,  195 

protozoa,  378 

soil  bacteria,  176 

Pathology,  relation  of  bacteriology  to,  7 
Pearl  disease  (bovine  tuberculosis),  310 
Pebrine,  10,  431,  432 

parasite  of,  431,  432* 

restriction  of,  432 
Penicillium  crustaceum,  234* 

glaucum,  138*,  234 
Peptone  solution,  92 
Peptonizing  ferments,  170 
Peritrichous  bacteria,  148 
Perlsucht  (bovine  tuberculosis),  310 
Permanganate  of  potassium,  74 
Peroxide  of  hydrogen,  74 
Pertussis,  296 
Petri  dishes,  108 
Pfeiffer's  phenomenon,  213 
Phagocytic  theory,  225,  227 
Phagocytosis,  207,  227 
Phenol,  76 

Phenolphthalein,  86,  87 
Phenomena  of  disease,  195 
Phenomenon,  Pfeiffer's,  213 
Phlebotomus  fever,  374 
Phosphorescence,  168 
Photogenic  bacteria,  168 
Phy  corny  cetes,  137 
Physical  sterilization,  62 
Physiological  method,  163 

hyperplasia,  206 

tests,  173 
Physiology  of  micro-organisms,  162 

relation  to  morphology,  162 
Pipettes,  glass  (Pasteur  pipette),  32,  33* 

for  drawing  blood  from  animal,  96* 

for  drawing  blood  from  man,  95* 
Piroplasma  (Babesia),  155,  158* 

bigeminum,  429* 

canis,  430 

muris,  158* 
Pityriasis,  242 
Placental  transmission,  198 
Plague,  317 

bubonic,  319 

diagnosis,  318 

fleas  as  carriers  of,  319 

Haffkine's  prophylactic,  320 

immunity,  320 

in  animals,  319,  320 

pneumonic,  320 

prophylaxis  of,  320,  321 

serum,  321 

transmission,  319,  320 


INDEX  OF  SUBJECTS 


457 


Plague  vaccines,  320 
Planococcus,  142,  143 

agilis,  267 

Planosarcina,  142,  143 
Plants,  diseases  of,  235 
Plasmodium,  12,  155,  157* 

brassicae,  407 
Plasmodium  falciparum,  157*,  420 

asexual  cycle  in  man,  420* 

cultures  of,  423 

pathogenic  relation  of,  426 

sexual  cycle  in  anopheles,  422* 

transmission  of,  423,  427 
Plasmodium  kochi,  429 

malariae,  425,  426* 

praecox,  420 

vivax,  424*,  425* 
Plasmodroma,  151 
Plasmolysis,  147 
Plate  cultures,  12,  106 

Koch's  original  method,  112 
Platinum  wire,  31,  32* 
Pleuro-pneumonia  of  cattle,  13,  368 

filterable  virus  of,  368 
Plugs,  cotton,  84 
Pneumococcus,  257,  258* 

immunity  to,  260 

poisons  of,  259 
Pneumonia,  259 

micro-organisms  in,  259 

serum,  260 
Poisoning,  food,  193,  284,  328 

botulism,  284 

enteritidis  type,  328 

proteus  vulgaris  as  cause  of,  343 
Poisons,  193,  203 
Poliomyelitis,  374 
Porcelain  filter,  63 

Post-mortem  examination,  98,  103,  136 
Postulates  of  Henle,  10,  1 2 

of  Koch,  12,  195 
Potassium  permanganate,  74 
Potato  cultures,  91 

bacillus  (B.  vulgatus),  268 

medium,  91* 
Precipitation  test,  209 
Precipitinogen,  210 
Precipitins,  209 
Predisposition,  197 
Preservation,  6,  62,  79 
Preservatives,  79 
Pressure,  effect  on  bacteria,  63 

filter,  63 
Products  of  bacteria,  168 

chemical  effects,  168 

enzymes,  169 

physical  effects,  168 


Products  of  primary,  169 

ptomaines,  170 

secondary,  169 

toxins,  171 
Protective  inoculation  for  anthrax,  273 

for  cholera,  351 

for  diphtheria,  295 

for  plague,  320 

for  small-pox,  376 

for  typhoid,  335 
Proteolysins,  218 
Proteosoma  praecox,  417 

development  in  blood,  418* 

development  in  the  mosquito,  419* 
Froteus     vulgaris    (Bacillus     proteus), 

343 

Protista,  160 
Protozoa,  12,  150 

relation  to  disease,  13 

wet  fixation  of,  54 
Pseudo-diphtheria  bacillus,  296 
Pseudomonas,  144 

radicicola,  175,  194 
Ptomain,  170 
Puerperal  fever,  263 
Pulmonary  anthrax,  273 
Pure  culture,  113 
Purification  of  water,  179 
Pus,  collection  of,  102 
Pustule,  malignant,  273 
Putrefaction,  5,  170 
Putrefactive  alkaloids,  170 

bacteria,  170 

products,  170 
Pyemia,  n,  200 
Pyoktanin,  78 
Pyrogallic-acid  anaerobic  method,  125 

Quartan  malaria,  425,  426*,  427 
Quarter    evil    (symptomatic    anthrax) 

276 

Quincke's  puncture,  255 
Quotidian  malaria,  426 

Rabies,  370,  372 

diagnosis  of,  372 
Hb'gyes  treatment  of,  222 
Negri  bodies  in,  370,  371* 
Pasteur  treatment,  373 
treatment  of  wound,  373 

Racial  immunity,  221 

Rats,  relation  to  bubonic  plague,  319, 

320,  321 
trypanosomes  01,  381 

Rauschbrand    (symptomatic    anthrax), 
276 

Ray  fungus  (actinomyces),  246,  247* 


458 


INDEX  OF  SUBJECTS 


Reaction,  cutaneous,  309 

of  culture  media,  165 

of  host  to  infection,  206 
Reading  glass,  19 
Receptor  of  first  order,  209* 

of  second  order,  210* 

of  third  order,  214* 

theory  of  immunity,  227 
Reducing  substances,  130 
Regulation  of  temperature,  115 
Regulator,  electric,  122* 

Roger's,  122*,  123 
Regulator,  gas,  116 

Mac  Neal,  117,  118* 

method  of  filling,  118 

Reichert,  117* 

Roux,  119* 
Relapsing  fever,  n,  353 

diagnosis,  355 

spirochetes,  353 
Resistance  to  infection,  196 
Respiratory  infection,  134 
Rhinoscleroma,  328 
Rhipicephalus  annul atus,  158,  429 
Rhizopoda,  151,  154*,  401 
Ricin,  171 

Rinderpest,  223,  370 
Roll-tubes,  no*,  in* 
Romano wsky  stain,  41,  43,  149 
Rooms,  disinfection  of,  72,  77 

incubator,  120 

Root-tubercle  bacteria,  14,  175,  194 
Rubber  caps,  114*,  115,  120 

stoppers,  114*,  115,  120 
Rules  of  Koch,  195 

Saccharomyces,  140 

cervisiae,  140*,  244 

ellipsoideus,  140* 
Sanarelli's  bacillus  (Bacillus  icteroides), 

329 

Sand  filtration,  180 
Saprogenic  bacteria,  170 
Saprophyte,  164 
Saprophytic,  164 
Sarcina,  142*,  143 

aurantiaca,  267 

ventriculi,  267 
Sarcoma,  chicken,  377 
Sarcoptes  scabei,  10 
Schizomycetes,  141 
Schizotrypanum  cruzi,  392,  393* 

cultures  of,  394 

transmission  of,  394 
Sealing  culture  tubes,  114*,  115,  120 
Secondary  infection,  199 
Section-cutting,  56 


Sections,  58 

staining  of,  58,  59 

tubercle  bacilli  in,  60 
Sedg wick-Tucker  aerobioscope,  177* 
Sedimentation,  63 
Self-purification  of  water,  179 
Semen,  transmission  of  infection  by,  198 
Sensitizer,  215 
•  Septicemia,  n,  200 

hemorrhagic,  316 

sputum,  277 
Serum,  anthrax,  274 

antibacterial,  212  * 

antimeningococcus,  255 

antipneumococcus,  260 

antistreptococcus,  264 

antitoxic,  208 

bactericidal,  212 

blood,  92 

cytolytic,  213 

dysentery,  337 

hemolytic,  216 

immune,  213 

Loffler's,  94 

normal,  217 

plague,  321 

Yersin's,  321 
Shiga's  bacillus   (Bacillus   dysenteriae) , 

336 

Side-chain  theory,  208,  227 
Silver  nitrate,  76 
Sleeping  sickness,  388,  390 

transmission  of,  388 

trypanosome  of,  388 

tsetse  fly  concerned  in,  388,  389* 
Slides,  forceps  for,  39 

glass,  39 

method  of  cleaning,  39 
Small -pox,  376 

inoculation,  12 

vaccination,  223,  376 

virus  of,  376 
Smear  culture,  cover-glass,  38 

preparations,  36 

slide,  39^ 

Smegma  bacilli,  314 
Soaps,  germicidal  action  of,  71 
Sodium  hydroxide,  normal  solution  of,  85 
Soft  chancre,  298 
Soil  bacteria,  175,  176 
Solutions,  normal,  85 
Soor  (thrush),  10,  238 
Sore,  Oriental  (Delhi  boil),  396 
Souring  of  milk,  191 
Species  of  bacteria,  167 

stability,  167 

variation,  167 


INDEX  OF  SUBJECTS 


459 


Specific  nomenclature,  160 
Sphaerophrya  pusilla,  437* 
Spherical  bacteria,  142* 
Spirilla,  142,  147*,  345 
Spirillacese,  146,  345 
Spirillum,  5,  142,  146 
Spirillum  choleras,  345 

agglutination,  350 

cultures  of,  345 

immunity,  348,  351 

in  feces,  350 

in  water,  351 

poisons  of,  348 

resistance  of,  346 

transmission  of,  349 
Spirillum,  Deneke's,  352 

metchnikovi,  352 

of  Finkler  and  Prior,  352 

rubrum,  345 

tyrogenum,  352 
Spirochaeta,  5,  13,  146 

anserina,  356 

culture  of,  13 

duttoni)>353,  354 

fusiformis,  367 

gallinarum,  356 

kochi,  354 

microdentium,  366 

muris,  356 

novyi,  354,  355* 

obermeieri,  353 

of  relapsing  fever,  353 
Spirochaeta  pallida,  357*,  359* 

animal  inoculation  of,  359 

antibodies,  361 

cultures  of,  357,  358 

in  blood,  361 

microscopic  demonstration  of,  360 

morphology,  357 

staining  of,  358 
Spirochaeta  plicatilis,  353 
Spirochaeta  recurrentis,  353 

transmission  of,  354,  355 

varieties  of,  353 
Spirochasta  refringens,  357*,  366 

suis,  374 

vincenti,  367 
Spirochetes,  5,  13,  146,  147,  353 

cultivation  of,  13 

of  mouth,  366 

of  relapsing  fevers,  353 

of  syphilis,  357* 

saprophytic,  353 
Spirosoma,  146 

Splenic  fever  (anthrax),  270,  272 
Splenomegaly,  tropical  (kala-azar),  394, 
396 


Spontaneous  generation,  3 
Sporogenic  aerobes,  268 

anaerobes,  275 
Spores,  146,  149*,  155 

formation  of,  145*,  149* 

germination  of,  149* 

resistance  of,  66 

staining  of,  50 
Sporotrichum  beurmanni,  244 

schencki,  242*,  243* 
Sporotrichosis,  242,  244 
Sporozoa,  151,  155 
Sporulation,  145* 
Sputum,  47 

collection  of,  47,  101 

examination  of,  48 

septicemia,  257 
Stab-culture,  113,  114*,  124 
Staining,  38,  44 

acid-proof  bacilli,  47 

anilin-water  gentian  violet,  40 

blood  films,  55 

capsules,  51 

dish,  39 

flagella,  52 

Gram's  method,  44 

Romano wsky,  41 

solutions,  39 

spirochetes,  358 

spores,  50 

tissues,  55 

tubercle  bacillus,  47,  49 
Standard  antitoxin,  282,  294 
Standardization  of  antitoxins,  281,  294 

diphtheria,  294 

tetanus,  281,  282 
Staphylococcus,  142*,  143,  264 

albus,  267 

aureus,  264 

epidermidis,  267 

immunity,  266 

infections,  264 

toxins,  265 

vaccines,  266 
Staphylolysin,  265 
Steam  sterilization,  66 

sterilizer,  66* 

Stegomyia  (Aedes)  calopus,  369* 
Sterilization,  62 

autoclave,  68 

by  desiccation,  63 

by  filtration,  63,  97 

by  light,  63 

by  moist  heat,  65 

by  sedimentation,  63 

chemical,  71 

fractional,  70 


460 


INDEX  OF  SUBJECTS 


Sterilization,  hot  air,  64 

mechanical,  62 

of  blood-serum,  93 

of  glassware,  84 

physical,  62 
Sterilizer,  Arnold,  67*,  68* 

hot-air,  65* 

Koch's,  for  serum,  93* 

steam,  66* 

Stewart's  forceps,  38* 
Stick-culture  (stab-culture),  114*,  124 
Stock  cultures,  114 
Stomach,  germicidal  action  of,  71 

infection  through,  198 
Stomoxys  calci trans,  387 
Stools  (feces),  102 
Straus's  test  for  glanders,  341 
Streak  cultures,  113,  114* 
Streptobacillus,  145 
Streptococcus,  142*,  143,  260 

erysipelas,  263 

erysipelatos,  260 

hemolytic,  261 

immunity,  264 

infections,  263 

lacticus,  190,  264 

mucosus,  260 

pyogenes,  260 

vaccines,  264 

viridans,  260 

virulence  of,  261 
Streptothrix,  246 

madurae,  248 

Structure  of  bacteria,  147 
Subcutaneous  application,  134 
Sugar-free  media,  90 
Sugars  in  culture  media,  90 
Sulphur  dioxide,  71 
Sunlight  as  germicide,  63 
Surra,  387  _ 
Susceptibility,  196 

local,  199 

Swine  erysipelas,  317 
Symbiosis,  194 
Symptomatic  anthrax,  276 
Syphilis,  357,  360 

diagnosis,  360,  366 

fixation  of  complement  in,  361,  366 

in  animals,  359,  361 

luetin  test  in,  366 

spirochete,  357,  360 

transmission  of,  360 

Wassermann  test  for,  361 
Systematic  relationships,  4,  137 

Telosporidia,  158 
Temperature,  influence  of,  167 


Temperature,  maximum,  166 

minimum,  166 

optimum,  166 

regulation  of,  115 

requirements,  166 

sterilizing,  68 
Tertian  malaria,  424*,  426 
Test,  Calmette's,  309 

complement- fixation,  216,  361 

luetin,  366 

mallei  n,  340 

precipitin,  210 

Straus's,  341 

tuberculin,  308,  311 

von  Pirquet's,  309 

Wassermann's,  361 

Testing  antiseptics  and  disinfectants,  80 
Tetanolysin,  279 
Tetanospasmin,  279 
Tetanus,  278,  280 

antitoxin,  281 

bacillus,  278,  280* 

immunity,  281 

immunity  unit,  281,  282 

prophylaxis,  283 

spasm  of,  280 

standard  antitoxin,  283 

toxin,  203,  279 

treatment  of,  283 
Tetrad,  142 
Texas  fever,  13,  429 

control  of,  430 

immunity  to,  223 

parasite  of,  429 

restriction  of,  430 

tick,  429 

Theories  of  immunity,  227 
Thermogenic  bacteria,  168 
Thermostat  (thermoregulator),  116,  122 
Thrush,  10,  238 
Tick,  cattle,  429 

fever,  354 
Tinea,  242 

versicolor  (pityriasis),  242 
Tissues,  examination  of,  55 
Titration  of  culture  media,  85,  87 
Tongue,  wooden  (actino mycosis),  246 
Torula,  141 
Toxemia,  200 
Toxin,  171,  203 

.  chemical  nature  of,  171 

diphtheria,  288 

extracellular,  203 

intracellular,  204 

soluble,  203 

standardization  of,  281,  294 

tetanus,  279 


INDEX  OF  SUBJECTS 


461 


Toxoid,  204 

Toxophore,  204 

Transmission  of  disease,  13,  197,  200 

Treponema  pallidum  (Spirochaeta  pal- 

lida),  357* 

Trichobacteria,  141,  246 
Trichomonas,  153,*  400 

hominis,  398,*  400 
Trichomycetes,  246 
Tricophyton,  242 
Trimastigamoeba  philippinensis,    399,* 

400 
Tropical  dysentery,  406 

malaria,  419,  426 

splenomegaly  (kala-azar),  394 

ulcer,  396 
Trypanoplasma  borreli,  398 

cyprini,  397,*  398 

guernei,  398 

Trypanosoma,  152,*  153,  379 
Trypanosoma  avium,  390,*  391* 

cultures  of,  392 

occurrence  of,  391 
Trypanosoma  brucei,  152,*  384* 

cultures  of,  384,  386 

form  and  structure,  384,  385 

immunity  to,  387 

multiplication  of,  386 

occurrence,  386 

poisons  of,  386 

transmission  of,  386 
Trypanosoma  equinum,  152,*  388 

equiperdum,  152,*  387* 

evansi,  152,*  387 
Trypanosoma  gambiense,  152,*  388 

cultures  of,  389 

form  and  structure,  388 

in  animals,  389 

in  man,  389 

in  the  fly,  388 

transmission  of,  388 
Trypanosoma  lewisi,  381* 

cultures  of,  383 

division  of,  382 

occurrence  of,  381 
Trypanosoma  rhodesiense,  390 

rotatorium,  379,  380* 
Trypanosomes,  152,*  379 
Tsetse-fly    (Glossina   morsitans),    385,* 
386 

disease  (Nagana),  384 
Tubercle,  306 
Tubercle  bacillus,  299 

amphibian,  312 

avian,  311 

bovine,  310 

branching  of,  302* 


Tubercle  bacillus,  chemical  composition 
of,  302 

cultures  of,  301 

fish  type,  312 

human  type,  300* 

in  sections,  60 

poisons  of,  303 

resistance  of,  304 

stain  for,  48 

transmission  of,  307 

varieties  of,  299 
Tuberculin,  304 

reaction,  308,  311 

test,  308,  309,  311 

treatment,  309 
Tuberculosis,  305 

avian,  311 

bacillus  of,  299 

bovine,  310 

diagnosis  of,  307 

fowl,  311 

immunity,  310 

mammalian,  299 

mode  of  infection  in,  307 

tuberculin  test  in,  308 

tuberculin  treatment  in,  310 
Typhoid  bacillus  (B.  typhosus),  330 

carriers,  333 

detection  in  water,  187 
Typhoid  fever,  333 

diagnosis  of,  333 

immunity  to,  335 

in  animals,  332 

prophylaxis  of,  335 

transmission  of,  333,  334 

vaccines,  335 

vaccination,  335 
Typhus  fever,  375 

Udder,  bacteria  in,  189 

Ulcer,  tropical,  396 

Ultramicroscope,  16 

Ultramicroscopic  organisms,  150,  368 

Unit,  immunity,  281,  294 

of  diphtheria  antitoxin,  294 
of  tetanus  antitoxin,  281,  282 

Urethritis,  252 

Urinary  bladder,  inflammation  of,  327 

Urine,  collection  of,  101 

Vaccination,  anthrax,  223 

Asiatic  cholera,  223 

small-pox,  12,  223,  376 

typhoid,  223 

Vaccines,  bacterial,  12,  223 
Vaccinia,  12 
Vagimtis,  gonorrheal,  252 


462 


INDEX  OF  SUBJECTS 


Van  Ermengem's  flagella  stain,  53 
Variola,  376 
Vibrio,  5 

choleras,  345 

Deneke's,  352 

metchnikovi,  352 

of  Finkler  and  Prior,  352 

tyrogenum,  352 

Vibrion  septique  (B.  edematis),  275 
Vincent's  angina,  367 

spirillum,  367 
Vinegar,  170 
Violet,  anilin-water  gentian,  40 

gentian,  40 

methyl  (pyoktanin),  78 

methylene.  43 
Virulence,  202 

factors  influencing,  202 

loss  of,  in  cultures,  115 
Virus,  filterable,  13,  150,  368 
Visibility  of  microscopic  objects,  25 

by  light  and  shade,  26*,  27* 

by  quality  of  light  (color),  28 
Von  Pirquet  test,  309 
Vulvo-vaginitis/  252 

Warmth,  115 
Wassermann  test,  361 
Water,  bacteria,  178,  183 

cholera  germs  in,  187 

collection  of  samples,  100 

disinfection  of,  182 

examination,  178 

filtration,  182 

intestinal  bacteria  in,  186 


Water,  self-purification  of   179 

storage  of,  180 

typhoid  bacilli  in,  187 
Watery  solution  of  aniline  dyes,  40 
Weigert's  stain,  59 
Welch's  bacillus  (B.  welchii),  276 

capsule  stain,  51 
Whooping  cough,  296 
Widal's  test  (agglutination),  211,  338 
Wire,  platinum,  31,  32* 
Wolff hiigel's  colony  counter,  185 
Wooden  tongue  (actino mycosis),  246 
Woolsorter's  -disease    (pulmonary    an- 
thrax), 273 
Wounds,  197 
Wright's  method  for  anaerobes,  125 

Xerosis  bacillus,  295 
Xylol,  55,  59 

Yeasts,  139,*  140* 
Yellow  fever,  368 

immunity,  369 

mosquito,  369* 

prophylaxis  of,  370 

transmission  of,  369 

virus,  368 
Yersin's  serum,  321 

Ziehl's  solution  (carbol-fuchsin),  46 
Zwischenkorper     (intermediary    body), 

214 

Zygospore,  137 
Zymogenic  bacteria,  169 
Zymophore,  211 


DATE    DUE    SLIP 

UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL  LIBRARY 


THIS  BOOK  IS  DUE   ON  THE   LAST   DATE 
STAMPED  BELOW 


MAY  7    1924 


$EP   3  U 

?9  1930 

5 

2  3  1931 
€0*21*31 


JAN   13   1932 

FEB  4      1932 
.SEP    ?«  ' 


MAR  '9-  1939 


2w-8,'21 


Library  of  the 
University  of  California  Medical  School  and  Hospitals 


